Δ12 desaturase gene suitable for altering levels of polyunsaturated fatty acids in oleaginous yeasts

ABSTRACT

The present invention relates to a Δ12 fatty acid desaturase able to catalyze the conversion of oleic acid to linoleic acid (LA; 18:2). Nucleic acid sequences encoding the desaturase, nucleic acid sequences that hybridize thereto, DNA constructs comprising the desaturase gene, and recombinant host microorganisms expressing increased levels of the desaturase are described. Methods of increasing production of specific ω-3 and/or ω-6 fatty acids are described by overexpression of the Δ12 fatty acid desaturase or by disruption of the native gene.

This application claims the benefit of U.S. Provisional Application No.60/468,677, filed May 7, 2003, and U.S. Provisional Application No.60/484,209, filed Jun. 30, 2003.

FIELD OF THE INVENTION

This invention is in the field of biotechnology. More specifically, thisinvention pertains to the identification of a nucleic acid fragmentencoding a Δ12 fatty acid desaturase enzyme useful for disrupting orenhancing the production of polyunsaturated fatty acids (PUFAs) inoleaginous microorganisms, such as oleaginous yeasts.

BACKGROUND OF THE INVENTION

It has long been recognized that certain polyunsaturated fatty acids, orPUFAs, are important biological components of healthy cells. Forexample, such PUFAs are recognized as:

-   -   “Essential” fatty acids that can not be synthesized de novo in        mammals and instead must be obtained either in the diet or        derived by further desaturation and elongation of linoleic acid        (LA) or α-linolenic acid (ALA);    -   Constituents of plasma membranes of cells, where they may be        found in such forms as phospholipids or triglycerides;    -   Necessary for proper development, particularly in the developing        infant brain, and for tissue formation and repair; and,    -   Precursors to several biologically active eicosanoids of        importance in mammals, including prostacyclins, eicosanoids,        leukotrienes and prostaglandins.

In the 1970's, observations of Greenland Eskimos linked a low incidenceof heart disease and a high intake of long-chain ω-3 PUFAs (Dyerberg, J.et al., Amer. J. Clin Nutr. 28:958-966 (1975); Dyerberg, J. et al.,Lancet 2(8081):117-119 (Jul. 15, 1978)). More recent studies haveconfirmed the cardiovascular protective effects of ω-3 PUFAs (Shimokawa,H., World Rev Nutr Diet, 88:100-108 (2001); von Schacky, C., andDyerberg, J., World Rev Nutr Diet, 88:90-99 (2001)). Further, it hasbeen discovered that several disorders respond to treatment with ω-3fatty acids, such as the rate of restenosis after angioplasty, symptomsof inflammation and rheumatoid arthritis, asthma, psoriasis and eczema.γ-linolenic acid (GLA, an ω-6 PUFA) has been shown to reduce increasesin blood pressure associated with stress and to improve performance onarithmetic tests. GLA and dihomo-γ-linolenic acid (DGLA, another ω-6PUFA) have been shown to inhibit platelet aggregation, causevasodilation, lower cholesterol levels and inhibit proliferation ofvessel wall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp.Med. Biol. 83: 85-101 (1976)). Administration of GLA or DGLA, alone orin combination with eicosapentaenoic acid (EPA, an ω-3 PUFA), has beenshown to reduce or prevent gastrointestinal bleeding and other sideeffects caused by non-steroidal anti-inflammatory drugs (U.S. Pat. No.4,666,701). Further, GLA and DGLA have been shown to prevent or treatendometriosis and premenstrual syndrome (U.S. Pat. No. 4,758,592) and totreat myalgic encephalomyelitis and chronic fatigue after viralinfections (U.S. Pat. No. 5,116,871). Other evidence indicates thatPUFAs may be involved in the regulation of calcium metabolism,suggesting that they may be useful in the treatment or prevention ofosteoporosis and kidney or urinary tract stones. Finally, PUFAs can beused in the treatment of cancer and diabetes (U.S. Pat. No. 4,826,877;Horrobin et al., Am. J. Clin. Nutr. 57 (Suppl.): 732S-737S (1993)).

PUFAs are generally divided into two major classes (consisting of theω-6 and the ω-3 fatty acids) that are derived by desaturation andelongation of the essential fatty acids, LA and ALA, respectively.Despite a variety of commercial sources of PUFAs from natural sources[e.g., seeds of evening primrose, borage and black currants; filamentousfungi (Mortierella), Porphyridium (red alga), fish oils and marineplankton (Cyclotella, Nitzschia, Crypthecodinium)], there are severaldisadvantages associated with these methods of production. First,natural sources such as fish and plants tend to have highlyheterogeneous oil compositions. The oils obtained from these sourcestherefore can require extensive purification to separate or enrich oneor more of the desired PUFAs. Natural sources are also subject touncontrollable fluctuations in availability (e.g., due to weather,disease, or over-fishing in the case of fish stocks); and, crops thatproduce PUFAs often are not competitive economically with hybrid cropsdeveloped for food production. Large-scale fermentation of someorganisms that naturally produce PUFAs (e.g., Porphyridium, Mortierella)can also be expensive and/or difficult to cultivate on a commercialscale.

As a result of the limitations described above, extensive work has beenconducted toward: 1.) the development of recombinant sources of PUFAsthat are easy to produce commercially; and 2.) modification of fattyacid biosynthetic pathways, to enable production of desired PUFAs. Forexample, advances in the isolation, cloning and manipulation of fattyacid desaturase and elongase genes from various organisms have been madeover the last several years. Knowledge of these gene sequences offersthe prospect of producing a desired fatty acid and/or fatty acidcomposition in novel host organisms that do not naturally produce PUFAs.The literature reports a number of examples in Saccharomyces cerevisiae,such as:

-   -   Domergue, F., et al. (Eur. J. Biochem. 269:4105-4113 (2002)),        wherein two desaturases from the marine diatom Phaeodactylum        tricornutum were cloned into S. cerevisiae, leading to the        production of EPA;    -   Beaudoin F., et al. (Proc. Natl. Acad. Sci. U.S.A.        97(12):6421-6426 (2000)), wherein the ω-3 and ω-6 PUF-A        biosynthetic pathways were reconstituted in S. cerevisiae, using        genes from Caenorhabditis elegans;    -   Dyer, J. M. et al. (Appl. Eniv. Microbiol., 59:224-230 (2002)),        wherein plant fatty acid desaturases (FAD2 and FAD3) were        expressed in S. cerevisiae, leading to the production of ALA;        and,    -   U.S. Pat. No. 6,136,574 (Knutzon et al., Abbott Laboratories),        wherein one desaturase from Brassica napus and two desaturases        from the fungus Mortierella alpina were cloned into S.        cerevisiae, leading to the production of LA, GLA, ALA and STA.        There remains a need, however, for an appropriate microbial        system in which these types of genes can be expressed to provide        for economical production of commercial quantities of one or        more PUFAs. Additionally, a need exists for oils enriched in        specific PUFAs, notably EPA and DHA.

One class or microorganisms that has not been previously examined as aproduction platform for PUFAs are the oleaginous yeasts. These organismscan accumulate oil up to 80% of their dry cell weight. The technologyfor growing oleaginous yeast with high oil content is well developed(for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol.16:119-206 (1982)) and may offer a cost advantage compared to commercialmicro-algae fermentation for production of ω-3 or ω-6 PUFAs. Whole yeastcells may also represent a convenient way of encapsulating ω-3 or ω-6PUFA-enriched oils for use in functional foods and animal feedsupplements.

Despite the advantages noted above, most oleaginous yeast are naturallydeficient in ω-6 and ω-3 PUFAs, since naturally produced PUFAs in theseorganisms are usually limited to 18:2 fatty acids (and less commonly,18:3 fatty acids). Thus, the problem to be solved is to develop anoleaginous yeast that accumulates oils enriched in ω-3 and/or ω-6 fattyacids. Toward this end, it is not only necessary to introduce therequired desaturases and elongases that allow for the synthesis andaccumulation of ω-3 and/or ω-6 fatty acids in oleaginous yeasts, butalso to increase the availability of the 18:2 substrate (i.e., LA).Generally, the availability of this substrate is controlled by theactivity of Δ12 desaturases that catalyze the conversion of oleic acidto LA.

There are a variety of known Δ12 desaturases disclosed in the publicliterature, some of which originate from fungal sources (e.g.,Mortierella alpina, Emericella nidulans, Mucor rouxil). Thesedesaturases are not known to be effective for altering fatty acidcomposition in oleaginous yeasts and are not preferred for use inoleaginous yeasts. Thus, there is need for the identification andisolation of genes encoding Δ12 desaturases that will be suitable forexpression in these particular host organisms for use in the productionof PUFAs.

Applicants have solved the stated problem by isolating the gene encodinga Δ12 desaturase from the oleaginous yeast, —Yarrowia lipolytica.

SUMMARY OF THE INVENTION

The invention relates to a gene encoding a Δ12 desaturase enzymeisolated from Yarrowia useful for the manipulation of the biochemicalpathway for the production of ω-3 and/or ω-6 fatty acids. Accordingly,the invention provides an isolated nucleic acid molecule encoding aYarrowia Δ12 desaturase enzyme, selected from the group consisting of:

-   -   (a) an isolated nucleic acid molecule encoding the amino acid        sequence as set forth in SEQ ID NO:24;    -   (b) an isolated nucleic acid molecule that hybridizes with (a)        under the following hybridization conditions: 0.1×SSC, 0.1% SDS,        65° C. and washed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1%        SDS; or an isolated nucleic acid molecule that is complementary        to (a) or (b).

Additionally the invention provides transformed host cells comprisingthe nucleic-acid molecules of the invention, genetic chimera andpolypeptides encoded by the same.

In an alternate embodiment the invention provides a method for theproduction of linoleic acid comprising:

-   -   a) providing a yeast comprising:        -   (i) a chimeric gene of the invention encoding a Δ12            desaturase polypeptide; and        -   (ii) a source of desaturase substrate consisting of oleic            acid;    -   b) growing the yeast of step (a) under conditions wherein the        gene encoding a Δ12 desaturase polypeptide is expressed and the        oleic acid is converted to linoleic acid; and    -   c) optionally recovering the linoleic acid of step (b).

In another embodiment the invention provides a method for producing ω-3fatty acids comprising:

-   -   a) engineering a microbial host cell comprising the following        elements:        -   (i) a disrupted endogenous gene encoding a Δ12 desaturase            polypeptide; and        -   (ii) genes encoding enzymes of the ω-3 fatty acid            biosynthetic pathway; and    -   b) providing a source of desaturase substrate consisting of        α-linolenic acid;    -   c) growing the yeast of step (a) under conditions wherein the        genes of the ω-3 fatty acid biosynthetic pathway are expressed,        producing ω-3 fatty acids; and    -   d) optionally recovering the ω-3 fatty acids of step (c).

Similarly the invention provides a method for modulating thebiosynthesis of ω-3 or ω-6 fatty acids in a host cell comprising:

-   -   a) providing a host cell comprising a functional ω-3/ω-6 fatty        acid biosynthetic pathway;    -   b) over-expressing a Δ12 desaturase gene in the host cell of        (a); whereby the biosynthesis of ω-3 or ω-6 fatty acids is        modulated.

In another embodiment the invention provides a method of obtaining anucleic acid molecule encoding a Δ12 desaturase enzyme comprising:

-   -   (a) probing a genomic library with the nucleic acid molecule of        the invention;    -   (b) identifying a DNA clone that hybridizes with the nucleic        acid molecule of the invention; and    -   (c) sequencing the genomic fragment that comprises the clone        identified in step (b),

wherein the sequenced genomic fragment encodes a Δ12 desaturase enzyme.

Similarly the invention provides a method of obtaining a nucleic acidmolecule encoding a Δ12 desaturase enzyme comprising:

-   -   (a) synthesizing at least one oligonucleotide primer        corresponding to a portion of the sequence as set forth in SEQ        ID NOs:23; and    -   (b) amplifying an insert present in a cloning vector using the        oligonucleotide primer of step (a);        wherein the amplified insert encodes a portion of an amino acid        sequence encoding a Δ12 desaturase enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE DESCRIPTIONS

FIG. 1 shows a schematic illustration of the biochemical mechanism forlipid accumulation in oleaginous yeast.

FIG. 2 illustrates the ω-3 and ω-6 fatty acid biosynthetic pathways.

FIG. 3 illustrates the construction of the plasmid vector pY5 for geneexpression in Yarrowia lipolytica.

FIG. 4 illustrates the construction of plasmid vectors pY5-13 and pY5-4for gene expression in Y. lipolytica.

FIG. 5 shows a pairwise comparison (% Identity) between and amongdifferent yeast and fungal Δ12 desaturase homologs using a ClustalWanalysis (Megalign program of DNASTAR sofware).

FIG. 6 is a schematic presentation of the construction of intermediatevector pYZM5CHPPA.

FIG. 7 shows a comparison between the DNA sequence of the Saprolegniadiclina Δ17 desaturase gene and the synthetic gene codon-optimized forexpression in Y. lipolytica.

FIG. 8 illustrates the favored consensus sequences around thetranslation initiation codon ‘ATG’ in Y. lipolytica.

FIG. 9 illustrates the strategy for in vitro synthesis of thecodon-optimized Δ17 desaturase gene.

FIG. 10 shows plasmids for expression of the synthetic codon-optimizedand wildtype Δ17 desaturase genes in Y. lipolytica.

FIGS. 11A and 11B show the results of gas chromatographic analysis offatty acids produced in Y. lipolytica transformed with the wildtype andsynthetic codon-optimized Δ17 desaturase genes, respectively.

FIG. 12 is a schematic presentation of the construction of intermediatevector pY24-4.

FIG. 13 is a schematic presentation of the construction of intermediatevector pYZV16.

FIG. 14 is a schematic presentation of the construction of integrationvector pYZM5EL6.

FIG. 15 is a schematic presentation of the construction of integrationvectors pYZV5EL6 and pYZV5EL6/17.

The invention can be more fully understood from the following detaileddescription and the accompanying sequence descriptions, which form apart of this application.

The following sequences comply with 37 C.F.R. §1.821-1.825(“Requirements for Patent Applications Containing Nucleotide Sequencesand/or Amino Acid Sequence Disclosures—the Sequence Rules”) and areconsistent with World Intellectual Property Organization (WIPO) StandardST.25 (1998) and the sequence listing requirements of the EPO and PCT(Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of theAdministrative Instructions). The symbols and format used for nucleotideand amino acid sequence data comply with the rules set forth in 37C.F.R. §1.822.

SEQ ID NOs:1 and 2 correspond to primers TEF5′ and TEF3′, respectively,used to isolate the TEF promoter.

SEQ ID NOs:3 and 4 correspond to primers XPR5′ and XPR3′, respectively,used to isolate the XPR2 transcriptional terminator.

SEQ ID NOs:5-18 correspond to primers YL1, YL2, YL3, YL4, YL23, YL24,YL5, YL6, YL9, YL10, YL7, YL8, YL61 and YL62, respectively, used forplasmid construction.

SEQ ID NOs:19 and 21 are the degenerate primers identified as P73 andP76, respectively, used for the isolation of a Yarrowia lioplytica Δ12desaturase gene.

SEQ ID NOs:20 and 22 are the amino acid consensus sequences thatcorrespond to the degenerate primers P73 and P76, respectively.

SEQ ID NO:23 shows the DNA sequence of the Y. lipolytica Δ12 desaturasegene, while SEQ ID NO:24 shows the amino acid sequence of the Y.lipolytica Δ2 desaturase.

SEQ ID NOs:25-28 correspond to primers P99, P100, P101 and P102,respectively, used for targeted disruption of the Y. lipolytica Δ12desaturase gene.

SEQ ID NOs:29-32 correspond to primers P119, P120, P121 and P122,respectively, used to screen for targeted integration of the disruptedY. lipolytica Δ12 desaturase gene.

SEQ ID NOs:33 and 34 correspond to primers P147 and P148, respectively,used to amplify the full-length Y. lipolytica Δ12 desaturase gene.

SEQ ID NO:35 shows the DNA sequence of the Saprolegnia diclina Δ17desaturase gene.

SEQ ID NO:36 shows the DNA sequence of the Mortierella alpina Δ6desaturase gene, while SEQ ID NO:37 shows the amino acid sequence of theM. alpina Δ6 desaturase.

SEQ ID NO:38 shows the DNA sequence of the Mortierella alpina Δ5desaturase gene, while SEQ ID NO:39 shows the amino acid sequence of theM. alpina Δ5 desaturase.

SEQ ID NOs:40 and 41 correspond to primers YL11 and YL12, respectively,used for amplifying the M. alpina Δ5 desaturase.

SEQ ID NOs:42 and 43 correspond to primers YL21A and YL22, respectively,used for amplifying the wild type S. diclina Δ17 desaturase.

SEQ ID NO:44 shows the DNA sequence of the Mortierella alpina highaffinity elongase gene, while SEQ ID NO:45 shows the amino acid sequenceof the M. alpina high affinity elongase.

SEQ ID NO:46 shows the DNA sequence of the synthetic Δ17 desaturase genecodon-optimized for expression in Yarrowia lipolytica, while SEQ IDNO:47 shows the corresponding amino acid sequence of the S. diclina Δ17desaturase.

SEQ ID NOs:48-69 correspond to the 11 pairs of oligonucleotides thattogether comprise the entire codon-optimized coding region of the S.diclina Δ17 desaturase gene (e.g., D17-1A, D17-1B, D17-2A, D17-2B,D17-3A, D17-3B, D17-4A, D17-4B, D17-5A, D17-5B, D17-6A, D17-6B, D17-7A,D17-7B, D17-8A, D17-8B, D17-9A, D17-9B, D17-10A, D17-10B, D17-11A andD17-11B, respectively).

SEQ ID NOs:70-75 correspond to primers D17-1, D17-4R, D17-5, D17-8D,D17-8U and D17-11, respectively, used for PCR amplification duringsynthesis of the codon-optimized Δ17 desaturase gene.

SEQ ID NOs:76 and 77 correspond to primers YL53 and YL54, respectively,used for site-directed mutagenesis to generate pYSD17M.

SEQ ID NOs:78 and 79 correspond to primers KU5 and KU3, respectively,used for amplifying a 1.7 kB DNA fragment (SEQ ID NO:80; amino acidsequence provided as SEQ ID NO:81) containing the Yarrowia URA3 gene.

SEQ ID NOs:82 and 83 correspond to primers KI5 and KI3, respectively,used for amplifying a 1.1 kB DNA fragment (SEQ ID NO:84; amino acidsequence provided as SEQ ID NO:85) containing the conjugase gene ofImpatients balsama.

SEQ ID NOs:86 and 87 correspond to primers KTI5 and KTI3, respectively,used for amplifying a 1.7 kB DNA fragment (SEQ ID NO:88; amino acidsequence provided as SEQ ID NO:89) containing a TEF::conjugase::XPRchimeric gene.

SEQ ID NOs:90 and 91 correspond to primers KH5 and KH3, respectively,used for amplifying a 1 kB DNA fragment (SEQ ID NO:92; amino acidsequence provided as SEQ ID NO:93) containing the E. coli hygromycinresistance gene.

SEQ ID NOs:94 and 95 correspond to primers KTH5 and KTH3, respectively,used for amplifying a 1.6 kB DNA fragment (SEQ ID NO:96; amino acidsequence provided as SEQ ID NO:97) containing the TEF::HPT::XPR fusiongene.

SEQ ID NOs:98 and 99 correspond to the 401 bp of 5′-sequence and 568 bpof 3′-sequence of the Yarrowia lipolytica URA3 gene, respectively, usedto direct integration of expression cassettes into the Ura loci of theYarrowia genome.

SEQ ID NOs:100-103 correspond to primers YL63, YL64, YL65 and YL66,respectively, used for site-directed mutagenesis to generate pY24-4.

SEQ ID NOs:104-107 correspond to primers YL81, YL82, YL83 and YL84,respectively, used for site-directed mutagenesis to generate pYZM5CH.

SEQ ID NOs:108 and 109 correspond to primers YL105 and YL106,respectively, used for site-directed mutagenesis to generate pYZM5CHPP.

SEQ ID NOs:110 and 111 correspond to primers YL119 and YL120,respectively, used for site-directed mutagenesis to generate pYZM5CHPPA.

SEQ ID NOs:112 and 113 correspond to primers YL121 and YL122,respectively, used for amplifying 440 bp of 5′-non-coding DNA sequence(SEQ ID NO:114) upstream from the Y. lipolylica URA3 gene.

SEQ ID NOs:115 and 116 correspond to primers YL114 and YL115,respectively, used for site-directed mutagenesis to generate pYZV5 andpYZV5P.

SEQ ID NO:117 corresponds to a 5.2 kB DNA fragment suitable forintegration and expression of the Δ5 desaturase gene in the Y.lipolytica genome.

SEQ ID NOs:118 and 119 correspond to primers YL69 and YL70,respectively, used for site-directed mutagenesis to generate pY58BH.

SEQ ID NOs:120-123-correspond to primers YL77, YL78, YL79A and YL80A,respectively, used for site-directed mutagenesis to generate pY54PC.

SEQ ID NO:124 corresponds to a 8.9 kB DNA fragment suitable forintegration and coordinate expression of the Δ6 desaturase, PUFAelongase and Δ5 desaturase genes in the Y. lipolytica genome.

SEQ ID NOs:125-128 correspond to primers YL101, YL102, YL103 and YL104,respectively, used for site-directed mutagenesis to generate pYSD17SPC.

SEQ ID NO:129 corresponds to a 10.3 kB DNA fragment suitable forintegration and coordinate expression of the Δ6 desaturase, PUFAelongase, Δ5 desaturase and Δ17 desaturase genes in the Y. lipolyticagenome.

SEQ ID NO:130 corresponds to the codon-optimized translation initiationsite for genes optimally expressed in Yarrowia sp.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, Applicants have isolated andconfirmed the identity of a Yarrowia lipolytica gene encoding a Δ12desaturase. Additionally, methods and compositions are provided whichpermit modification of the long chain polyunsaturated fatty acid (PUFA)content of oleaginous yeasts, such as Yarrowia lipolytica.

The invention relates to a new Δ12 desaturase enzyme and gene encodingthe same that may be used for the manipulation of biochemical pathwaysfor the production of healthful PUFAs. The subject invention finds manyapplications. PUFAs, or derivatives thereof, made by the methodologydisclosed herein can be used as dietary substitutes, or supplements,particularly infant formulas, for patients undergoing intravenousfeeding or for preventing or treating malnutrition. Alternatively, thepurified PUFAs (or derivatives thereof) may be incorporated into cookingoils, fats or margarines formulated so that in normal use the recipientwould receive the desired amount for dietary supplementation. The PUFAsmay also be incorporated into infant formulas, nutritional supplementsor other food products and may find use as anti-inflammatory orcholesterol lowering agents. Optionally, the compositions may be usedfor pharmaceutical use (human or veterinary). In this case, the PUFAsare generally administered orally but can be administered by any routeby which they may be successfully absorbed, e.g., parenterally (e.g.,subcutaneously, intramuscularly or intravenously), rectally, vaginallyor topically (e.g., as a skin ointment or lotion).

Supplementation of humans or animals with PUFAs produced by recombinantmeans can result in increased levels of the added PUFAs, as well astheir metabolic progeny. For example, treatment with arachidonic acid(ARA) can result not only in increased levels of ARA, but alsodownstream products of ARA such as prostaglandins. Complex regulatorymechanisms can make it desirable to combine various PUFAs, or adddifferent conjugates of PUFAs, in order to prevent, control or overcomesuch mechanisms to achieve the desired levels of specific PUFAs in anindividual.

Definitions

In this disclosure, a number of terms and abbreviations are used. Thefollowing definitions are provided.

“Open reading frame” is abbreviated ORF.

“Polymerase chain reaction” is abbreviated PCR.

“American Type Culture Collection” is abbreviated ATCC.

“Polyunsaturated fatty acid(s)” is abbreviated PUFA(s).

The term “fatty acids” refers to long chain aliphatic acids (alkanoicacids) of varying chain length, from about C₁₂ to C₂₂ (although bothlonger and shorter chain-length acids are known). The predominant chainlengths are between C₁₆ and C₂₂. The structure of a fatty acid isrepresented by a simple notation system of “X:Y”, where X is the totalnumber of carbon (C) atoms in the particular fatty acid and Y is thenumber of double bonds.

Generally, fatty acids are classified as saturated or unsaturated. Theterm “saturated fatty acids” refers to those fatty acids that have no“double bonds” between their carbon backbone. In contrast, “unsaturatedfatty acids” have “double bonds” along their carbon backbones (which aremost commonly in the cis-configuration). “Monounsaturated fatty acids”have only one “double bond” along the carbon backbone (e.g., usuallybetween the 9^(th) and 10^(th) carbon atom as for palmitoleic acid(16:1) and oleic acid (18:1)), while “polyunsaturated fatty acids” (or“PUFAs”) have at least two double bonds along the carbon backbone (e.g.,between the 9^(th) and 10^(th), and 12^(th) and 13^(th) carbon atoms forlinoleic acid (18:2); and between the 9^(th) and 10^(th), 12^(th) and13^(th), and 15^(th) and 16^(th) for α-linolenic acid (18:3)).

“PUFAs” can be classified into two major families (depending on theposition (n) of the first double bond nearest the methyl end of thefatty acid carbon chain). Thus, the “omega-6 fatty acids” (ω-6 or n-6)have the first unsaturated double bond six carbon atoms from the omega(methyl) end of the molecule and additionally have a total of two ormore double bonds, with each subsequent unsaturation occuring 3additional carbon atoms toward the carboxyl end of the molecule. Incontrast, the “omega-3 fatty acids” ω-3 or n-3) have the firstunsaturated double bond three carbon atoms away from the omega end ofthe molecule and additionally have a total of three or more doublebonds, with each subsequent unsaturation occuring 3 additional carbonatoms toward the carboxyl end of the molecule.

For the purposes of the present disclosure, the omega-reference systemwill be used to indicate the number of carbons, the number of doublebonds and the position of the double bond closest to the omega carbon,counting from the omega carbon (which is numbered 1 for this purpose).This nomenclature is shown below in Table 1, in the column titled“Shorthand Notation”. The remainder of the Table summarizes the commonnames of ω-3 and ω-6 fatty acids, the abbreviations that will be usedthroughout the specification and each compounds' chemical name.

TABLE 1 Nomenclature Of Polyunsaturated Fatty Acids Shorthand CommonName Abbreviation Chemical Name Notation Linoleic LAcis-9,12-octadecadienoic 18:2 ω-6 γ-Linoleic GLA cis-6,9,12- 18:3 ω-6octadecatrienoic Dihomo-γ- DGLA cis-8,11,14- 20:3 ω-6 Linoleiceicosatrienoic Arachidonic ARA cis-5,8,11,14- 20:4 ω-6 eicosatetraenoicα-Linolenic ALA cis-9,12,15- 18:3 ω-3 octadecatrienoic Stearidonic STAcis-6,9,12,15- 18:4 ω-3 octadecatetraenoic Eicosa- ETA cis-8,11,14,17-20:4 ω-3 tetraenoic eicosatetraenoic Eicosa- EPA cis-5,8,11,14,17- 20:5ω-3 pentaenoic eicosapentaenoic Docosa- DPA cis-7,10,13,16,19- 22:5 ω-3pentaenoic docosapentaenoic Docosa- DHA cis-4,7,10,13,16,19- 22:6 ω-3hexaenoic docosahexaenoic

The term “essential fatty acid” refers to a particular PUFA that anindividual must ingest in order to survive, being unable to synthesizethe particular essential fatty acid de novo. Linoleic (18:2, ω-6) andlinolenic (18:3, ω-3) fatty acids are “essential fatty acids”, sincehumans cannot synthesize them and have to obtain them in their diet.

The term “fat” refers to a lipid substance that is solid at 25° C. andusually saturated.

The term “oil” refers to a lipid substance that is liquid at 25° C. andusually polyunsaturated. PUFAs are found in the oils of some algae,oleaginous yeasts and filamentous fungi. “Microbial oils” or “singlecell oils” are those oils naturally produced by microorganisms duringtheir lifespan. Such oils can contain long chain PUFAs.

The term “PUFA biosynthetic pathway enzyme” refers to any of thefollowing enzymes (and genes which encode said enzymes) associated withthe biosynthesis of a PUFA, including: a Δ4 desaturase, a Δ5 desaturase,a Δ6 desaturase, a Δ12 desaturase, a Δ15 desaturase, a Δ17 desaturase, aΔ9 desaturase and/or an elongase.

The term “ω-3/ω-6 fatty acid biosynthetic pathway” refers to a set ofgenes which, when expressed under the appropriate conditions encodeenzymes that catalyze the production of either or both ω-3 and ω-6 fattyacids. Typically the genes involved in the ω-3/ω-6 fatty acidbiosynthetic pathway encode some or all of the following enzymes:Δ12desaturase, Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase, Δ15desaturase, Δ9 desaturase and Δ4 desaturase. A representative pathway isillustrated in FIG. 2, providing for the conversion of oleic acidthrough various intermediates to DHA, which demonstrates how both ω-3and ω-6 fatty acids may be produced from a common source. The pathway isnaturally divided into two portions where one portion will generate ω-3fatty acids and the other portion, only ω-6 fatty acids. That portionthat only generates ω-3 fatty acids will be referred to herein as theω-3 fatty acid biosynthetic pathway whereas that portion that generatesonly ω-6 fatty acids will be referred to herein as the ω-6 fatty acidbiosynthetic pathway.

The term “functional” as used herein in context with the ω-3/ω-6 fattyacid biosynthetic pathway means that some or all of the genes in thepathway express active enzymes. It should be understood that “ω-3/ω-6fatty acid biosynthetic pathway” or “functional ω-3/ω-6 fatty acidbiosynthetic pathway” does not imply that all the genes listed in thisparagraph are required as a number of fatty acid products will onlyrequire the expression of a subset of the genes of this pathway.

The term “desaturase” refers to a polypeptide that can desaturate, i.e.,introduce a double bond, in one or more fatty acids to produce a mono-or polyunsaturated fatty acid. Despite use of the omega-reference systemthroughout the specification in reference to specific fatty acids, it ismore convenient to indicate the activity of a desaturase by countingfrom the carboxyl end of the substrate using the delta-system. Ofparticular interest herein are Δ12 desaturases that desaturate a fattyacid between the 12^(th) and 13^(th) carbon atoms numbered from thecarboxyl-terminal end of the molecule and that catalyze the conversionof oleic acid to LA. Other desaturases relevant to the presentdisclosure include: Δ15 desaturases that catalyze the conversion of LAto ALA; Δ17 desaturases that desaturate a fatty acid between the 17^(th)and 18^(th) carbon atom numbered from the carboxyl-terminal end of themolecule and which, for example, catalyze the conversion of ARA to EPAand/or DGLA to ETA; Δ6 desaturases that catalyze the conversion of LA toGLA and/or ALA to STA; Δ5 desaturases that catalyze the conversion ofDGLA to ARA and/or ETA to EPA; Δ4 desaturases that catalyze theconversion of DPA to DHA; and Δ9 desaturases that catalyze theconversion of palmitate to palmitoleic acid (16:1) and/or stearate tooleic acid (18:1).

The term “elongase” refers to a polypeptide that can elongate a fattyacid carbon chain to produce an acid that is 2 carbons longer than thefatty acid substrate that the elongase acts upon. This process ofelongation occurs in a multi-step mechanism in association with fattyacid synthase, whereby CoA is the acyl carrier (Lassner et al., ThePlant Cell 8:281-292 (1996)). Briefly, malonyl-CoA is condensed with along-chain acyl-CoA to yield CO₂ and a β-ketoacyl-CoA (where the acylmoiety has been elongated by two carbon atoms). Subsequent reactionsinclude reduction to β-hydroxyacyl-CoA, dehydration to an enoyl-CoA anda second reduction to yield the elongated acyl-CoA. Examples ofreactions catalyzed by elongases are the conversion of GLA to DGLA, STAto ETA and EPA to DPA. Accordingly, elongases can have differentspecificities (e.g., a C_(16/18) elongase will prefer a C₁₆ substrate, aC_(18/20) elongase will prefer a C₁₈ substrate and a C_(20/22) elongasewill prefer a C₂₀ substrate).

The terms “conversion efficiency” and “percent substrate conversion”refer to the efficiency by which a particular enzyme (e.g., a desaturaseor elongase) can convert substrate to product. The conversion efficiencyis measured according to the following formula:([product]/[substrate+product])*100, where ‘product’ includes theimmediate product and all products in the pathway derived from it.

The term “oleaginous” refers to those organisms that tend to store theirenergy source in the form of lipid (Weete, In: Fungal LipidBiochemistry, 2^(nd) ed., Plenum, 1980). Generally, the cellular oil ortriacylglycerol content of oleaginous microorganisms follows a sigmoidcurve, wherein the concentration of lipid increases until it reaches amaximum at the late logarithmic or early stationary growth phase andthen gradually decreases during the late stationary and death phases(Yongmanitchai and Ward, Appl. Environ. Microbiol. 57:419-25 (1991)).

The term “oleaginous yeast” refers to those microorganisms classified asyeasts that can accumulate at least 25% of their dry cell weight as oil.Examples of oleaginous yeast include, but are no means limited to, thefollowing genera: Yarrowia, Candida, Rhodotorula, Rhodosporidium,Cryptococcus, Trichosporon and Lipomyces.

The term “fermentable carbon substrate” means a carbon source that amicroorganism will metabolize to derive energy. Typical carbonsubstrates of the invention include, but are not limited to:monosaccharides, oligosaccharides, polysaccharides, alkanes, fattyacids, esters of fatty acids, monoglycerides, carbon dioxide, methanol,formaldehyde, formate and carbon-containing amines.

The term “codon-optimized” as it refers to genes or coding regions ofnucleic acid molecules for transformation of various hosts, refers tothe alteration of codons in the gene or coding regions of the nucleicacid molecules to reflect the typical codon usage of the host organismwithout altering the polypeptide encoded by the DNA.

As used herein, an “isolated nucleic acid fragment” is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. An isolated nucleicacid fragment in the form of a polymer of DNA may be comprised of one ormore segments of cDNA, genomic DNA or synthetic DNA.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA molecule, when asingle-stranded form of the nucleic acid molecule can anneal to theother nucleic acid molecule under the appropriate conditions oftemperature and solution ionic strength. Hybridization and washingconditions are well known and exemplified in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989),particularly Chapter 11 and Table 11.1 therein (entirely incorporatedherein by reference). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. Stringency conditionscan be adjusted to screen for moderately similar fragments (such ashomologous sequences from distantly related organisms), to highlysimilar fragments (such as genes that duplicate functional enzymes fromclosely related organisms). Post-hybridization washes determinestringency conditions. One set of preferred conditions uses a series ofwashes starting with 6×SSC, 0.5% SDS at room temperature for 15 min,then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and thenrepeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A morepreferred set of stringent conditions uses higher temperatures in whichthe washes are identical to those above except for the temperature ofthe final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C.Another preferred set of highly stringent conditions uses two finalwashes in 0.1×SSC, 0.1% SDS at 65° C. An additional set of stringentconditions include hybridization at 0.1×SSC, 0.1% SDS, 65° C. and washedwith 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS, for example.

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridizations with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). In one embodimentthe length for a hybridizable nucleic acid is at least about 10nucleotides. Preferably a minimum length for a hybridizable nucleic acidis at least about 15 nucleotides; more preferably at least about 20nucleotides; and most preferably the length is at least about 30nucleotides. Furthermore, the skilled artisan will recognize that thetemperature and wash solution salt concentration may be adjusted asnecessary according to factors such as length of the probe.

A “substantial portion” of an amino acid or nucleotide sequence is thatportion comprising enough of the amino acid sequence of a polypeptide orthe nucleotide sequence of a gene to putatively identify thatpolypeptide or gene, either by manual evaluation of the sequence by oneskilled in the art, or by computer-automated sequence comparison andidentification using algorithms such as BLAST (Basic Local AlignmentSearch Tool; Altschul, S. F., et al., J. Mol. Biol. 215:403-410 (1993).In general, a sequence of ten or more contiguous amino acids or thirtyor more nucleotides is necessary in order to putatively identify apolypeptide or nucleic acid sequence as homologous to a known protein orgene. Moreover, with respect to nucleotide sequences, gene specificoligonucleotide probes comprising 20-30 contiguous nucleotides may beused in sequence-dependent methods of gene identification (e.g.,Southern hybridization) and isolation (e.g., in situ hybridization ofbacterial colonies or bacteriophage plaques). In addition, shortoligonucleotides of 12-15 bases may be used as amplification primers inPCR in order to obtain a particular nucleic acid fragment comprising theprimers. Accordingly, a “substantial portion” of a nucleotide sequencecomprises enough of the sequence to specifically identify and/or isolatea nucleic acid fragment comprising the sequence. The instantspecification teaches the complete amino acid and nucleotide sequenceencoding a particular yeast protein. The skilled artisan, having thebenefit of the sequences as reported herein, may now use all or asubstantial portion of the disclosed sequences for purposes known tothose skilled in this art. Accordingly, the instant invention comprisesthe complete sequences as reported in the accompanying Sequence Listing,as well as substantial portions of those sequences as defined above.

The term “complementary” is used to describe the relationship betweennucleotide bases that are capable of hybridizing to one another. Forexample, with respect to DNA, adenosine is complementary to thymine andcytosine is complementary to guanine. Accordingly, the instant inventionalso includes isolated nucleic acid fragments that are complementary tothe complete sequences as reported in the accompanying Sequence Listing,as well as those substantially similar nucleic acid sequences.

The term “percent identity”, as known in the art, is a relationshipbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,“identity” also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. “Identity”and “similarity” can be readily calculated by known methods, includingbut not limited to those described in: 1.) Computational MolecularBiology (Lesk, A. M., Ed.) Oxford University: NY (1988); 2.)Biocomputing: Informatics and Genome Projects (Smith, D. W., Ed.)Academic: NY (1993); 3.) Computer Analysis of Sequence Data, Part I(Griffin, A. M., and Griffin, H. G., Eds.) Humana: NJ (1994); 4.)Sequence Analysis in Molecular Biology (von Heinje, G., Ed.) Academic(1987); and 5.) Sequence Analysis Primer (Gribskov, M. and Devereux, J.,Eds.) Stockton: NY (1991). Preferred methods to determine identity aredesigned to give the best match between the sequences tested. Methods todetermine identity and similarity are codified in publicly availablecomputer programs. Sequence alignments and percent identity calculationsmay be performed using the Megalign program of the LASERGENEbioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiplealignment of the sequences is performed using the Clustal method ofalignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments using the Clustal method are: KTUPLE 1, GAPPENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

Suitable nucleic acid fragments (isolated polynucleotides of the presentinvention) encode polypeptides that are at least about 70% identical,preferably at least about 75% identical, and more preferably at leastabout 80% identical to the amino acid sequence reported herein.Preferred nucleic acid fragments encode amino acid sequences that areabout 85% identical to the amino acid sequence reported herein. Morepreferred nucleic acid fragments encode amino acid sequences that are atleast about 90% identical to the amino acid sequence reported herein.Most preferred are nucleic acid fragments that encode amino acidsequences that are at least about 95% identical to the amino acidsequence reported herein. Suitable nucleic acid fragments not only havethe above homologies but typically encode a polypeptide having at least50 amino acids, preferably at least 100 amino acids, more preferably atleast 150 amino acids, still more preferably at least 200 amino acids,and most preferably at least 250 amino acids.

“Codon degeneracy” refers to the nature in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a gene for improved expression in a host cell, it isdesirable to design the gene such that its frequency of codon usageapproaches the frequency of preferred codon usage of the host cell.

“Chemically synthesized”, as related to a sequence of DNA, means thatthe component nucleotides were assembled in vitro. Manual chemicalsynthesis of DNA may be accomplished using well-established procedures;or automated chemical synthesis can be performed using one of a numberof commercially available machines. “Synthetic genes” can be assembledfrom oligonucleotide building blocks that are chemically synthesizedusing procedures known to those skilled in the art. These buildingblocks are ligated and annealed to form gene segments that are thenenzymatically assembled to construct the entire gene. Accordingly, thegenes can be tailored for optimal gene expression based on optimizationof nucleotide sequence to reflect the codon bias of the host cell. Theskilled artisan appreciates the likelihood of successful gene expressionif codon usage is biased towards those codons favored by the host.Determination of preferred codons can be based on a survey of genesderived from the host cell, where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specificprotein, including regulatory sequences preceding (5′ non-codingsequences) and following (3′ non-coding sequences) the coding sequence.“Native gene” refers to a gene as found in nature with its ownregulatory sequences. “Chimeric gene” refers to any gene that is not anative gene, comprising regulatory and coding sequences that are notfound together in nature. Accordingly, a chimeric gene may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than that foundin nature. “Endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism, but that is introduced into thehost organism by gene transfer. Foreign genes can comprise native genesinserted into a non-native organism, or chimeric genes. A “transgene” isa gene that has been introduced into the genome by a transformationprocedure. A “codon-optimized gene” is a gene having its frequency ofcodon usage designed to mimic the frequency of preferred codon usage ofthe host cell.

“Coding sequence” refers to a DNA sequence that codes for a specificamino acid sequence. “Suitable regulatory sequences” refer to nucleotidesequences located upstream (5′ non-coding sequences), within ordownstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences may includepromoters., translation leader sequences, introns, polyadenylationrecognition sequences, RNA processing sites, effector binding sites andstem-loop structures.

“Promoter” refers to a DNA sequence capable of controlling theexpression of a coding sequence or functional RNA. In general, a codingsequence is located 3′ to a promoter sequence. Promoters may be derivedin their entirety from a native gene, or be composed of differentelements derived from different promoters found in nature, or evencomprise synthetic DNA segments. It is understood by those skilled inthe art that different promoters may direct the expression of a gene indifferent tissues or cell types, or at different stages of development,or in response to different environmental or physiological conditions.Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”. It isfurther recognized that since in most cases the exact boundaries ofregulatory sequences have not been completely defined, DNA fragments ofdifferent lengths may have identical promoter activity.

The term “3′ non-coding sequences” or “transcription terminator” refersto DNA sequences located downstream of a coding sequence. This includespolyadenylation recognition sequences and other sequences encodingregulatory signals capable of affecting mRNA processing or geneexpression. The polyadenylation signal is usually characterized byaffecting the addition of polyadenylic acid tracts to the 3′ end of themRNA precursor. The 3′ region can influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence.

“RNA transcript” refers to the product resulting from RNApolymerase-catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from post-transcriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” or “mRNA” refersto the RNA that is without introns and that can be translated intoprotein by the cell. “cDNA” refers to a double-stranded DNA that iscomplementary to, and derived from, mRNA. “Sense” RNA refers to RNAtranscript that includes the mRNA and so can be translated into proteinby the cell. “Antisense RNA” refers to a RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target gene (U.S. Pat. No. 5,107,065; WO99/28508). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, or the coding sequence. “Functional RNA” refers toantisense RNA, ribozyme RNA, or other RNA that is not translated and yethas an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acidsequences on a single nucleic acid fragment so that the function of oneis affected by the other. For example, a promoter is operably linkedwith a coding sequence when it is capable of affecting the expression ofthat coding sequence (i.e., the coding sequence is under thetranscriptional control of the promoter). Coding sequences can beoperably linked to regulatory sequences in sense or antisenseorientation.

The term “expression”, as used herein, refers to the transcription andstable accumulation of sense (mRNA) or antisense RNA derived from thenucleic acid fragment(s) of the invention. Expression may also refer totranslation of mRNA into a polypeptide.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or propeptides present in the primarytranslation product have been removed. “Precursor” protein refers to theprimary product of translation of mRNA; i.e., with pre- and propeptidesstill present. Pre- and propeptides may be (but are not limited to)intracellular localization signals.

“Transformation” refers to the transfer of a nucleic acid molecule intoa host organism, resulting in genetically stable inheritance. Thenucleic acid molecule may be a plasmid that replicates autonomously, forexample; or, it may integrate into the genome of the host organism. Hostorganisms containing the transformed nucleic acid fragments are referredto as “transgenic” or “recombinant” or “transformed” organisms.

The terms “plasmid”, “vector” and “cassette” refer to an extrachromosomal element often carrying genes that are not part of thecentral metabolism of the cell, and usually in the form of circulardouble-stranded DNA fragments. Such elements may be autonomouslyreplicating sequences, genome integrating sequences, phage or nucleotidesequences, linear or circular, of a single- or double-stranded DNA orRNA, derived from any source, in which a number of nucleotide sequenceshave been joined or recombined into a unique construction which iscapable of introducing a promoter fragment and DNA sequence for aselected gene product along with appropriate 3′ untranslated sequenceinto a cell. “Transformation cassette” refers to a specific vectorcontaining a foreign gene and having elements in addition to the foreigngene that facilitate transformation of a particular host cell.“Expression cassette” refers to a specific vector containing a foreigngene and having elements in addition to the foreign gene that allow forenhanced expression of that gene in a foreign host.

The term “altered biological activity” will refer to an activity,associated with a protein encoded by a nucleotide sequence which can bemeasured by an assay method, where that activity is either greater thanor less than the activity associated with the native sequence. “Enhancedbiological activity” refers to an altered activity that is greater thanthat associated with the native sequence. “Diminished biologicalactivity” is an altered activity that is less than that associated withthe native sequence.

The term “homologous recombination” refers to the exchange of DNAfragments between two DNA molecules (during cross over). The fragmentsthat are exchanged are flanked by sites of identical nucleotidesequences between the two DNA molecules (i.e., “regions of homology”).The term “regions of homology” refer to stretches of nucleotide sequenceon nucleic acid fragments that participate in homologous recombinationthat have homology to each other. Effective homologous recombinationwill take place where these regions of homology are at least about 10 bpin length where at least about 50 bp in length is preferred. Typicallyfragments that are intended for recombination contain at least tworegions of homology where targeted gene disruption or replacement isdesired.

The term “sequence analysis software” refers to any computer algorithmor software program that is useful for the analysis of nucleotide oramino acid sequences. “Sequence analysis software” may be commerciallyavailable or independently developed. Typical sequence analysis softwarewill include, but is not limited to: 1.) the GCG suite of programs(Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison,Wis.); 2.) BLASTP, BLASTN, BLASTX (Altschul et al., J. Mol. Biol.215:403-410 (1990)); 3.) DNASTAR (DNASTAR, Inc. Madison, Wis.); 4.)Sequencher (Gene Codes Corporation, Ann Arbor, Mich.); and 5.) the FASTAprogram incorporating the Smith-Waterman algorithm (W. R. Pearson,Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date1992, 111-20. Editor(s): Suhai, Sandor. Plenum: New York, N.Y.). Withinthe context of this application it will be understood that wheresequence analysis software is used for analysis, that the results of theanalysis will be based on the “default values” of the programreferenced, unless otherwise specified. As used herein “default values”will mean any set of values or parameters that originally load with thesoftware when first initialized.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(hereinafter “Maniatis”); by Silhavy, T. J., Bennan, M. L. and Enquist,L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory:Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Microbial Biosynthesis of Fatty Acids

In general, lipid accumulation in oleaginous microorganisms is triggeredin response to the overall carbon to nitrogen ratio present in thegrowth medium (FIG. 1). When cells have exhausted available nitrogensupplies (e.g., when the carbon to nitrogen ratio is greater than about40), the depletion of cellular adenosine monophosphate (AMP) leads tothe cessation of AMP-dependent isocitrate dehydrogenase activity in themitochondria and the accumulation of citrate, transport of citrate intothe cytosol and subsequent cleavage of the citrate by ATP-citrate lyaseto yield acetyl-CoA. Acetyl-CoA is the principle building block for denovo biosynthesis of fatty acids. Although any compound that caneffectively be metabolized to produce acetyl-CoA can serve as aprecursor of fatty acids, glucose is the primary source of carbon inthis type of reaction (FIG. 1). Glucose is converted to pyruvate viaglycolysis and pyruvate is then transported into the mitochondria whereit can be converted to acetyl-CoA by pyruvate dehydrogenase (“PD”).Since acetyl-CoA can not be transported directly across themitochondrial membrane into the cytoplasm, the two carbons fromacetyl-CoA condense with oxaloacetate to yield citrate (catalyzed bycitrate synthase). Citrate is transported directly into the cytoplasm,where it is cleaved by ATP-citrate lyase to regenerate acetyl-CoA andoxaloacetate. The oxaloacetate reenters the tricarboxylic acid cycle,via conversion to malate.

The synthesis of malonyl-CoA is the first committed step of fatty acidbiosynthesis, which takes place in the cytoplasm. Malonyl-CoA isproduced via carboxylation of acetyl-CoA by acetyl-CoA carboxylase(“ACC”). Fatty acid synthesis is catalyzed by a multi-enzyme fatty acidsynthase complex (“FAS”) and occurs by the condensation of eighttwo-carbon fragments (acetyl groups from acetyl-CoA) to form a 16-carbonsaturated fatty acid, palmitate. More specifically, FAS catalyzes aseries of 7 reactions, which involve the following (Smith, S. FASEB J,8(15):1248-59 (1994)):

-   -   1. Acetyl-CoA and malonyl-CoA are transferred to the acyl        carrier peptide (ACP) of FAS. The acetyl group is then        transferred to the malonyl group, forming β-ketobutyryl-ACP and        releasing CO₂.    -   2. The β-ketobutyryl-ACP undergoes reduction (via β-ketoacyl        reductase) and dehydration (via β-hydroxyacyl dehydratase) to        form a trans-monounsaturated fatty acyl group.    -   3. The double bond is reduced by NADPH, yielding a saturated        fatty-acyl group two carbons longer than the initial one. The        butyryl-group's ability to condense with a new malonyl group and        repeat the elongation process is then regenerated.    -   4. When the fatty acyl group becomes 16 carbons long, a        thioesterase activity hydrolyses it, releasing free palmitate.

Palmitate (16:0) is the precursor of longer chain saturated andunsaturated fatty acids (e.g., stearic (18:0), palmitoleic (16:1) andoleic (18:1) acids) through the action of elongases and desaturasespresent in the endoplasmic reticulum membrane. Palmitate and stearateare converted to their unsaturated derivatives, palmitoleic (16:1) andoleic (18:1) acids, respectively, by the action of a Δ9 desaturase.

Triacylglycerols (the primary storage unit for fatty acids) are formedby the esterification of two molecules of acyl-CoA toglycerol-3-phosphate to yield 1,2-diacylglycerol phosphate (commonlyidentified as phosphatidic acid) (FIG. 1). The phosphate is thenremoved, by phosphatidic acid phosphatase, to yield 1,2-diacylglycerol.Triacylglycerol is formed upon the addition of a third fatty acid, forexample, by the action of a diacylglycerol-acyl transferase.

Biosynthesis of Omega Fatty Acids

Simplistically, the metabolic process that converts LA to GLA, DGLA andARA (the ω-6 pathway) and ALA to STA, ETA, EPA, DPA and DHA (the ω-3pathway) involves elongation of the carbon chain through the addition oftwo-carbon units and desaturation of the molecule through the additionof double bonds (FIG. 2). This requires a series of special desaturationand elongation enzymes present in the endoplasmic reticulum membrane.

ω-6 Fatty Acids

Oleic acid is converted to LA (18:2), the first of the ω-6 fatty acids,by the action of a Δ12 desaturase. Subsequent ω-6 fatty acids areproduced as follows: 1.) LA is converted to GLA by the activity of a Δ6desaturase; 2.) GLA is converted to DGLA by the action of an elongase;and 3.) DGLA is converted to ARA by the action of a Δ5 desaturase.

ω-3 Fatty Acids

Linoleic acid (LA) is converted to ALA, the first of the ω-3 fattyacids, by the action of a Δ15 desaturase. Subsequent ω-3 fatty acids areproduced in a series of steps similar to that for the ω-6 fatty acids.Specifically: 1.) ALA is converted to STA by the activity of a Δ6desaturase; 2.) STA is converted to ETA by the activity of an elongase;and 3.) ETA is converted to EPA by the activity of a Δ5 desaturase.Alternatively, ETA and EPA can be produced from DGLA and ARA,respectively, by the activity of a Δ17 desaturase. EPA can be furtherconverted to DHA by the activity of an elongase and a Δ4 desaturase.

Genes Involved in Omega Fatty Acid Production

Many microorganisms, including algae, bacteria, molds and yeasts, cansynthesize PUFAs and omega fatty acids in the ordinary course ofcellular metabolism. Particularly well-studied are fungi includingSchizochytrium aggregatm, species of the genus Thraustochytrium andMorteriella alpina. Additionally, many dinoflagellates (Dinophyceaae)naturally produce high concentrations of PUFAs. As such, a variety ofgenes involved in oil production have been identified through geneticmeans and the DNA sequences of some of these genes are publiclyavailable (non-limiting examples are shown below in Table 2):

TABLE 2 Some Publicly Available Genes Involved in PUFA ProductionGenbank Accession No. Description AY131238 Argania spinosa Δ6 desaturaseY055118 Echium pitardii var. pitardii Δ6 desaturase AY055117 Echiumgentianoides Δ6 desaturase AF296076 Mucor rouxii Δ6 desaturase AF007561Borago officinalis Δ6 desaturase L11421 Synechocystis sp. Δ6 desaturaseNM_031344 Rattus norvegicus Δ6 fatty acid desaturase AF465283,Mortierella alpina Δ6 fatty acid desaturase AF465281, AF110510 AF465282Mortierella isabellina Δ6 fatty acid desaturase AF419296 Pythiumirregulare Δ6 fatty acid desaturase AB052086 Mucor circinelloides D6dmRNA for Δ6 fatty acid desaturase AJ250735 Ceratodon purpureus mRNA forΔ6 fatty acid desaturase AF126799 Homo sapiens Δ6 fatty acid desaturaseAF126798 Mus musculus Δ6 fatty acid desaturase AF199596, Homo sapiens Δ5desaturase AF226273 AF320509 Rattus norvegicus liver Δ5 desaturaseAB072976 Mus musculus D5D mRNA for Δ5 desaturase AF489588Thraustochytrium sp. ATCC21685 Δ5 fatty acid desaturase AJ510244Phytophthora megasperma mRNA for Δ5 fatty acid desaturase AF419297Pythium irregulare Δ5 fatty acid desaturase AF07879 Caenorhabditiselegans Δ5 fatty acid desaturase AF067654 Mortierella alpina Δ5 fattyacid desaturase AB022097 Dictyostelium discoideum mRNA for Δ5 fatty aciddesaturase AF489589.1 Thraustochytrium sp. ATCC21685 Δ4 fatty aciddesaturase AX464731 Mortierella alpina elongase gene (also WO 00/12720)AAG36933 Emericella nidulans oleate Δ12 desaturase AF110509 Mortierellaalpina Δ12 fatty acid desaturase mRNA AB020033 Mortierella alpina mRNAfor Δ12 fatty acid desaturase AAL13300 Mortierella alpina Δ12 fatty aciddesaturase AF417244 Mortierella alpina ATCC 16266 Δ12 fatty aciddesaturase gene AF161219 Mucor rouxii Δ12 desaturase mRNA X86736Spiruline platensis Δ12 desaturase AF240777 Caenorhabditis elegans Δ12desaturase AB007640 Chlamydomonas reinhardtii Δ12 desaturase AB075526Chlorella vulgaris Δ12 desaturase AP002063 Arabidopsis thalianamicrosomal Δ12 desaturase AY332747 Pavlova lutheri Δ4 fatty aciddesaturase (des1) mRNA NP_441622, Synechocystis sp. PCC 6803 Δ15desaturase BAA18302, BAA02924 AAL36934 Perilla frutescens Δ15 desaturaseAF338466 Acheta domesticus Δ9 desaturase 3 mRNA AF438199 Picea glaucadesaturase Δ9 (Des9) mRNA E11368 Anabaena Δ9 desaturase E11367Synechocystis Δ9 desaturase D83185 Pichia angusta DNA for Δ9 fatty aciddesaturase U90417 Synechococcus vulcanus Δ9 acyl-lipid fatty aciddesaturase (desC) gene AF085500 Mortierella alpina Δ9 desaturase mRNAAY504633 Emericella nidulans Δ9 stearic acid desaturase (sdeB) geneNM_069854 Caenorhabditis elegans essential fatty acid desatur- ase,stearoyl-CoA desaturase (39.1 kD) (fat-6) complete mRNA AF230693Brassica oleracea cultivar Rapid Cycling stearoyl-ACP desaturase(Δ9-BO-1) gene, exon sequence AX464731 Mortierella alpina elongase gene(also WO 02/08401) NM_119617 Arabidopsis thaliana fatty acid elongase 1(FAE1) (At4g34520) mRNA NM_134255 Mus musculus ELOVL family member 5,elongation of long chain fatty acids (yeast) (Elovl5), mRNA NM_134383Rattus norvegicus fatty acid elongase 2 (rELO2), mRNA NM_134382 Rattusnorvegicus fatty acid elongase 1 (rELO1), mRNA NM_068396, Caenorhabditiselegans fatty acid ELOngation (elo-6), NM_068392, (elo-5), (elo-2),(elo-3), and (elo-9) mRNA NM_070713, NM_068746, NM_064685

Additionally, the patent literature provides many additional DNAsequences of genes (and/or details concerning several of the genes aboveand their methods of isolation) involved in PUFA production. See, forexample: U.S. Pat. No. 5,968,809 (Δ6 desaturases); U.S. Pat. No.5,972,664 and U.S. Pat. No. 6,075,183 (Δ5 desaturases); WO 91/13972 andU.S. Pat. No. 5,057,419 (Δ9 desaturases); WO 93/11245 (Δ15 desaturases);U.S. 2003/0196217 A1 (Δ17 desaturases), WO 02/090493 (Δ4 desaturases);and WO 00/12720 and U.S. 2002/0139974A1 (elongases). Each of thesepatents and applications are herein incorporated by reference in theirentirety.

Of particular interest herein are Δ12 desaturases, and morespecifically, Δ12 desaturases that are suitable for expression inoleaginous yeast (e.g., Yarrowia lipolytica). A variety of sequencesencoding fungal Δ12 fatty acid desaturases have been previouslydisclosed that could be used for heterologous expression in oleaginousYarrowia lipolytica (e.g., GenBank Accession No's MG36933, AF110509,AAL13300, AF417244, AF161219 (supra)). Additionally, for example, theΔ12 fatty acid desaturases of Glycine max, Brassica napus, Arabidopsisthaliana, Ricinus communis, Zea mays; Neurospora crassa and Botrytiscinerea are disclosed in WO 94/11516, U.S. Pat. No. 5,443,974 and WO03/099216.

Many factors affect the choice of a specific polypeptide having Δ12desaturase activity that is to be expressed in a host cell forproduction of PUFAs (optionally in combination with other desaturasesand elongases). Depending upon the host cell, the availability ofsubstrate and the desired end product(s), several polypeptides are ofinterest; however, considerations for choosing a specific polypeptidehaving desaturase activity include the substrate specificity of thepolypeptide, whether the polypeptide or a component thereof is arate-limiting enzyme, whether the desaturase is essential for synthesisof a desired polyunsaturated fatty acid and/or co-factors required bythe polypeptide. The expressed polypeptide preferably has parameterscompatible with the biochemical environment of its location in the hostcell. For example, the polypeptide may have to compete for substratewith other enzymes in the host cell. Analyses of the K_(M) and specificactivity of the polypeptide are therefore considered in determining thesuitability of a given polypeptide for modifying PUFA production in agiven host cell. The polypeptide used in a particular host cell is onethat can function under the biochemical conditions present in theintended host cell, but otherwise can be any polypeptide having Δ12desaturase activity capable of modifying the desired fatty acid (i.e.,oleic acid). Thus, the sequences may be derived from any source, e.g.,isolated from a natural source (from bacteria, algae, fungi, plants,animals, etc.), produced via a semi-synthetic route or synthesized denovo.

Sequence Identification of the Yarrowia lipolytica Δ12 Desaturase

Despite public disclosure of a variety of sequences encoding fungal Δ12fatty acid desaturases (supra), expression of a native enzyme ispreferred over a heterologous (or “foreign”) enzyme since: 1.) thenative enzyme is optimized for interaction with other enzymes andproteins within the cell; and 2.) heterologous genes are unlikely toshare the same codon preference in the host organism. Additionally,advantages are incurred when the sequence of the native gene is known,as it permits facile disruption of the endogenous gene by targeteddisruption.

Concerning disruption of a native Δ12 fatty acid desaturase gene, it maybe useful for to engineer an oleaginous yeast that is not capable ofproducing PUFAs in some embodiments. Commercial applications where thislack of functionality would be desirable include the production of highvalue cocoa butter substitutes, oxidatively stable oils and specialtyfatty acids derived from 18:1 (e.g., hydroxy- and epoxy-fatty acids).Alternatively, oleaginous yeast lacking Δ12 fatty acid desaturaseactivity could be utilized to produce “pure” ω-3 derivatives of ALA(e.g., STA, ETA, EPA, DPA, DHA) by transforming the organism with theappropriate genes (e.g., Δ6 desaturase, elongase, Δ5 desaturase, Δ4desaturase) and feeding the organism ALA as a substrate; ω-6 fatty acidswould not be synthesized under these conditions (see FIG. 2).

Thus, the Applicants sought to isolate a Δ12 fatty acid desaturase fromYarrowia lipolytica. Comparison of the Δ12 desaturase nucleotide baseand deduced amino acid sequences to public databases reveals that themost similar known sequences are about 53% identical to the amino acidsequence of Δ12 desaturase reported herein (SEQ ID NO:24) over a lengthof 419 amino acids using a Clustal method of alignment (Thompson et.al., Nucleic Acids Res. 22:4673-4680 (1994)). More preferred amino acidfragments are at least about 70%-80% identical to the sequence herein,where those sequences that are 85%-90% identical are particularlysuitable and those sequences that are about 95% identical are mostpreferred. Similarly, preferred Δ12 desaturase encoding nucleic acidsequences corresponding to the instant ORF are those encoding activeproteins and which are at least about 70%-80% identical to the nucleicacid sequence of Δ12 desaturase reported herein, where those sequencesthat are 85%-90% identical are particularly suitable and those sequencesthat are about 95% identical are most preferred.

Isolation Of Homologs

The Δ12 desaturase nucleic acid fragment of the instant invention may beused to isolate genes encoding homologous proteins from the same orother bacterial, algal, fungal or plant species. Isolation of homologousgenes using sequence-dependent protocols is well known in the art.Examples of sequence-dependent protocols include, but are not limitedto: 1.) methods of nucleic acid hybridization; 2.) methods of DNA andRNA amplification, as exemplified by various uses of nucleic acidamplification technologies [e.g., polymerase chain reaction (PCR),Mullis et al., U.S. Pat. No. 4,683,202; ligase chain reaction (LCR),Tabor, S. et al., Proc. Acad. Sci. USA 82:1074 (1985); or stranddisplacement amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci.U.S.A., 89:392 (1992)]; and 3.) methods of library construction andscreening by complementation.

For example, genes encoding similar proteins or polypeptides to thedesaturase described herein could be isolated directly by using all or aportion of the instant nucleic acid fragments as DNA hybridizationprobes to screen libraries from any desired yeast or fungus usingmethodology well known to those skilled in the art (wherein those yeastor fungus producing LA and/or LA-derivatives would be preferred).Specific oligonucleotide probes based upon the instant nucleic acidsequences can be designed and synthesized by methods known in the art(Maniatis, supra). Moreover, the entire sequences can be used directlyto synthesize DNA probes by methods known to the skilled artisan (e.g.,random primers DNA labeling, nick translation or end-labelingtechniques) or RNA probes using available in vitro transcriptionsystems. In addition, specific primers can be designed and used toamplify a part of (or full-length of) the instant sequences. Theresulting amplification products can be labeled directly duringamplification reactions or labeled after amplification reactions, andused as probes to isolate full-length DNA fragments under conditions ofappropriate stringency.

Typically, in PCR-type amplification techniques, the primers havedifferent sequences and are not complementary to each other. Dependingon the desired test conditions, the sequences of the primers should bedesigned to provide for both efficient and faithful replication of thetarget nucleic acid. Methods of PCR primer design are common and wellknown in the art (Thein and Wallace, “The use of oligonucleotide asspecific hybridization probes in the Diagnosis of Genetic Disorders”, inHuman Genetic Diseases: A Practical Approach, K. E. Davis Ed., (1986) pp33-50, IRL: Herndon, Va.; and Rychlik, W., In Methods in MolecularBiology, White, B. A. Ed., (1993) Vol. 15, pp 31-39, PCR Protocols:Current Methods and Applications. Humania: Totowa, N.J.).

Generally two short segments of the instant sequences may be used inpolymerase chain reaction protocols to amplify longer nucleic acidfragments encoding homologous genes from DNA or RNA. The polymerasechain reaction may also be performed on a library of cloned nucleic acidfragments wherein the sequence of one primer is derived from the instantnucleic acid fragments and the sequence of the other primer takesadvantage of the presence of the polyadenylic acid tracts to the 3′ endof the mRNA precursor encoding microbial genes.

Alternatively, the second primer sequence may be based upon sequencesderived from the cloning vector. For example, the skilled artisan canfollow the RACE protocol (Frohman et al., PNAS USA 85:8998 (1988)) togenerate cDNAs by using PCR to amplify copies of the region between asingle point in the transcript and the 3′ or 5′ end. Primers oriented inthe 3′ and 5′ directions can be designed from the instant sequences.Using commercially available 3′ RACE or 5′ RACE systems (BRL,Gaithersburg, Md.), specific 3′ or 5′ cDNA fragments can be isolated(Ohara et al., PNAS USA 86:5673 (1989); Loh et al., Science 243:217(1989)).

In other embodiments, the instant desaturase sequences may be employedas hybridization reagents for the identification of homologs. The basiccomponents of a nucleic acid hybridization test include a probe, asample suspected of containing the gene or gene fragment of interest anda specific hybridization method. Probes of the present invention aretypically single-stranded nucleic acid sequences that are complementaryto the nucleic acid sequences to be detected. Probes are “hybridizable”to the nucleic acid sequence to be detected. The probe length can varyfrom 5 bases to tens of thousands of bases, and will depend upon thespecific test to be done. Typically a probe length of about 15 bases toabout 30 bases is suitable. Only part of the probe molecule need becomplementary to the nucleic acid sequence to be detected. In addition,the complementarity between the probe and the target sequence need notbe perfect. Hybridization does occur between imperfectly complementarymolecules with the result that a certain fraction of the bases in thehybridized region are not paired with the proper complementary base.

Hybridization methods are well defined. Typically the probe and samplemust be mixed under conditions that will permit nucleic acidhybridization. This involves contacting the probe and sample in thepresence of an inorganic or organic salt under the proper concentrationand temperature conditions. The probe and sample nucleic acids must bein contact for a long enough time that any possible hybridizationbetween the probe and sample nucleic acid may occur. The concentrationof probe or target in the mixture will determine the time necessary forhybridization to occur. The higher the probe or target concentration,the shorter the hybridization incubation time needed. Optionally, achaotropic agent may be added. The chaotropic agent stabilizes nucleicacids by inhibiting nuclease activity. Furthermore, the chaotropic agentallows sensitive and stringent hybridization of short oligonucleotideprobes at room temperature (Van Ness and Chen, Nucl. Acids Res.19:5143-5151 (1991)). Suitable chaotropic agents include guanidiniumchloride, guanidinium thiocyanate, sodium thiocyanate, lithiumtetrachloroacetate, sodium perchlorate, rubidium tetrachloroacetate,potassium iodide and cesium trifluoroacetate, among others. Typically,the chaotropic agent will be present at a final concentration of about 3M. If desired, one can add formamide to the hybridization mixture,typically 30-50% (v/v).

Various hybridization solutions can be employed. Typically, thesecomprise from about 20 to 60% volume, preferably 30%, of a polar organicsolvent. A common hybridization solution employs about 30-50% v/vformamide, about 0.15 to 1 M sodium chloride, about 0.05 to 0.1 Mbuffers (e.g., sodium citrate, Tris-HCl, PIPES or HEPES (pH range about6-9)), about 0.05 to 0.2% detergent (e.g., sodium dodecylsulfate), orbetween 0.5-20 mM EDTA, FICOLL (Pharmacia Inc.) (about 300-500 kdal),polyvinylpyrrolidone (about 250-500 kdal) and serum albumin. Alsoincluded in the typical hybridization solution will be unlabeled carriernucleic acids from about 0.1 to 5 mg/mL, fragmented nucleic DNA (e.g.,calf thymus or salmon sperm DNA, or yeast RNA), and optionally fromabout 0.5 to 2% wt/vol glycine. Other additives may also be included,such as volume exclusion agents that include a variety of polarwater-soluble or swellable agents (e.g., polyethylene glycol), anionicpolymers (e.g., polyacrylate or polymethylacrylate) and anionicsaccharidic polymers (e.g., dextran sulfate).

Nucleic acid hybridization is adaptable to a variety of assay formats.One of the most suitable is the sandwich assay format. The sandwichassay is particularly adaptable to hybridization under non-denaturingconditions. A primary component of a sandwich-type assay is a solidsupport. The solid support has adsorbed to it or covalently coupled toit immobilized nucleic acid probe that is unlabeled and complementary toone portion of the sequence.

Availability of the instant nucleotide and deduced amino acid sequencesfacilitates immunological screening of DNA expression libraries.Synthetic peptides representing portions of the instant amino acidsequence may be synthesized. These peptides can be used to immunizeanimals to produce polyclonal or monoclonal antibodies with specificityfor peptides or proteins comprising the amino acid sequences. Theseantibodies can be then be used to screen DNA expression libraries toisolate full-length DNA clones of interest (Lerner, R. A. Adv. Immunol.36:1 (1984); Maniatis, supra).

Gene Optimization for Improved Heterologous Expression

A variety of techniques can be utilized to improve the expression of theΔ12 desaturase in an alternate host. Two such techniques includecodon-optimization and mutagenesis of the gene.

Codon Optimization

In some embodiments, it may be desirable to modify a portion of thecodons encoding the Δ12 desaturase polypeptide, for example, to enhancethe expression of the gene encoding that polypeptide in an alternatehost (e.g., an oleaginous yeast other than Yarrowia lipolytica).

In general, host preferred codons can be determined within a particularhost species of interest by examining codon usage in proteins(preferably those proteins expressed in the largest amount) anddetermining which codons are used with highest frequency. Then, thecoding sequence for the polypeptide of interest having desaturaseactivity can be synthesized in whole or in part using the codonspreferred in the host species. All (or portions) of the DNA also can besynthesized to remove any destabilizing sequences or regions ofsecondary structure that would be present in the transcribed mRNA. All(or portions) of the DNA also can be synthesized to alter the basecomposition to one more preferable in the desired host cell.

Mutagenesis

Methods for synthesizing sequences and bringing sequences together arewell established in the literature. For example, in vitro mutagenesisand selection, site-directed mutagenesis, error prone PCR (Melnikov etal., Nucleic Acids Research, 27(4):1056-1062 (Feb. 15, 1999)), “geneshuffling” (U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; and5,837,458) or other means can be employed to obtain mutations ofnaturally occurring desaturase genes, such as the Δ12 desaturasedescribed herein. This would permit production of a polypeptide havingdesaturase activity in vivo with more desirable physical and kineticparameters for function in the host cell (e.g., a longer half-life or ahigher rate of production of a desired PUFA).

If desired, the regions of a desaturase polypeptide important forenzymatic activity can be determined through routine mutagenesis,expression of the resulting mutant polypeptides and determination oftheir activities. Mutants may include deletions, insertions and pointmutations, or combinations thereof. A typical functional analysis beginswith deletion mutagenesis to determine the N- and C-terminal limits ofthe protein necessary for function, and then internal deletions,insertions or point mutants are made to further determine regionsnecessary for function. Other techniques such as cassette mutagenesis ortotal synthesis also can be used. Deletion mutagenesis is accomplished,for example, by using exonucleases to sequentially remove the 5′ or 3′coding regions. Kits are available for such techniques. After deletion,the coding region is completed by ligating oligonucleotides containingstart or stop codons to the deleted coding region after the 5′ or 3′deletion, respectively. Alternatively, oligonucleotides encoding startor stop codons are inserted into the coding region by a variety ofmethods including site-directed mutagenesis, mutagenic PCR or byligation onto DNA digested at existing restriction sites. Internaldeletions can similarly be made through a variety of methods includingthe use of existing restriction sites in the DNA, by use of mutagenicprimers via site-directed mutagenesis or mutagenic PCR. Insertions aremade through methods such as linker-scanning mutagenesis, site-directedmutagenesis or mutagenic PCR. Point mutations are made throughtechniques such as site-directed mutagenesis or mutagenic PCR.

Chemical mutagenesis also can be used for identifying regions of adesaturase polypeptide important for activity. A mutated construct isexpressed, and the ability of the resulting altered protein to functionas a desaturase is assayed. Such structure-function analysis candetermine which regions may be deleted, which regions tolerateinsertions, and which point mutations allow the mutant protein tofunction in substantially the same way as the native desaturase. Allsuch mutant proteins and nucleotide sequences encoding them that arederived from the desaturase described herein are within the scope of thepresent invention.

Thus, the present invention comprises the complete sequence of the Δ12desaturase as reported in the accompanying Sequence Listing, thecomplement of that complete sequence, substantial portions of thatsequence, codon-optimized desaturases derived therefrom and thosesequences that are substantially homologous thereto.

Microbial Production of ω-3 and/or ω-6 Fatty Acids

Microbial production of ω-3 and/or ω-6 fatty acids has severaladvantages over purification from natural sources such as fish orplants. For example:

-   -   1.) Many microbes are known with greatly simplified oil        compositions compared with those of higher organisms, making        purification of desired components easier;    -   2.) Microbial production is not subject to fluctuations caused        by external variables, such as weather and food supply;    -   3.) Microbially produced oil is substantially free of        contamination by environmental pollutants;    -   4.) Microbes can provide PUFAs in particular forms which may        have specific uses; and    -   5.) Microbial oil production can be manipulated by controlling        culture conditions, notably by providing particular substrates        for microbially expressed enzymes, or by addition of compounds        or genetic engineering approaches to suppress undesired        biochemical pathways.        In addition to these advantages, production of ω-3 and/or ω-6        fatty acids from recombinant microbes provides the ability to        alter the naturally occurring microbial fatty acid profile by        providing new biosynthetic pathways in the host or by        suppressing undesired pathways, thereby increasing levels of        desired PUFAs (or conjugated forms thereof) and decreasing        levels of undesired PUFAs (see co-pending U.S. Provisional        Application 60/468,677, herein incorporated entirely by        reference).

Methods for Production of Various ω-3 and/or ω-6 Fatty Acids

It is expected that introduction of chimeric genes encoding the Δ12desaturase described herein, under the control of appropriate promoterswill result in increased production of LA. As such, the presentinvention encompasses a method for the direct production of PUFAscomprising exposing a fatty acid substrate (i.e., oleic acid) to thePUFA enzyme described herein (i.e., the Δ12 desaturase), such that thesubstrate is converted to the desired fatty acid product (i.e., LA).

Alternatively, the PUFA gene and its corresponding enzyme productdescribed herein can be used indirectly for the production of PUFAs.Indirect production of PUFAs occurs wherein the fatty acid substrate isconverted indirectly into the desired fatty acid product, via means ofan intermediate step(s) or pathway intermediate(s). Thus, it iscontemplated that the Δ12 desaturase described herein may be expressedin conjunction with one or more genes that encode other enzymes, suchthat a series of reactions occur to produce a desired product. In apreferred embodiment, for example, a host organism may be co-transformedwith a vector comprising additional genes encoding enzymes of the PUFAbiosynthetic pathway to result in higher levels of production of ω-3and/or ω-6 fatty acids (e.g., GLA, DGLA, ARA, ALA, STA, ETA, EPA, DPAand DNA). Specifically, for example, it may be desirable to overexpressthe Δ12 desaturase described herein in host cells that are alsoexpressing: 1.) a gene encoding a Δ6 desaturase for the overproductionof GLA; 2.) an expression cassette comprising genes encoding a Δ6desaturase and a high-affinity elongase for the overproduction of DGLA;3.) genes encoding a Δ6 desaturase, high-affinity elongase and Δ5desaturase for the overproduction of ARA; or 4.) genes encoding a Δ6desaturase, high-affinity elongase, Δ5 desaturase and Δ17 desaturase forthe overproduction of EPA. In alternate embodiments, it may be desirableto overexpress the Δ12 desaturase as described herein in cells that arealso expressing: 1.) a gene encoding a Δ15 desaturase for theoverproduction of ALA; 2.) genes encoding a Δ15 desaturase and Δ6desaturase for the overproduction of STA; 3.) genes encoding a Δ15desaturase, Δ16 desaturase and a high-affinity elongase for theoverproduction of ETA; or 4.) genes encoding a Δ15 desaturase, Δ6desaturase, high-affinity elongase and Δ5 desaturase for theoverproduction of EPA. As is well known to one skilled in the art,various other combinations of the following enzymatic activities may beuseful to express in a host in conjunction with the desaturase herein: aΔ15 desaturase, a Δ4 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ17desaturase, a Δ9 desaturase and/or an elongase (see FIG. 2). Theparticular genes included within a particular expression cassette willdepend on the host cell (and its PUFA profile and/or desaturaseprofile), the availability of substrate and the desired end product(s).

In alternate embodiments, it may be useful to disrupt a host organism'snative Δ12 desaturase, based on the complete sequences described herein,the complement of those complete sequences, substantial portions ofthose sequences, codon-optimized desaturases derived therefrom and thosesequences that are substantially homologous thereto. For example, thetargeted disruption of the Δ12 desaturase described herein in Yarrowialipolytica produces a mutant strain that is unable to synthesize LA.This mutant strain could be useful for: 1.) production of otherspecialty oils (e.g., high value cocoa butter substitutes, oxidativelystable oils and fatty acids derived from 18:1 such as hydroxy- andepoxy-fatty acids); or 2.) production of “pure” ω-3 fatty acidderivatives of ALA, when the host cells are grown on e.g., ALA (withoutco-synthesis of ω-6 fatty acids).

Expression Systems, Cassettes and Vectors

The gene and gene product of the instant sequences described herein maybe produced in various microbial host cells, particularly in the cellsof oleaginous yeasts (e.g., Yarrowia lipolytica). Expression inrecombinant microbial hosts may be useful for the production of variousPUFA pathway intermediates, or for the modulation of PUFA pathwaysalready existing in the host for the synthesis of new productsheretofore not possible using the host.

Microbial expression systems and expression vectors containingregulatory sequences that direct high level expression of foreignproteins are well known to those skilled in the art. Any of these couldbe used to construct chimeric genes for production of any of the geneproducts of the instant sequences. These chimeric genes could then beintroduced into appropriate microorganisms via transformation to providehigh-level expression of the encoded enzymes.

Vectors or DNA cassettes useful for the transformation of suitable hostcells are well known in the art. The specific choice of sequencespresent in the construct is dependent upon the desired expressionproducts (supra), the nature of the host cell and the proposed means ofseparating transformed cells versus non-transformed cells. Typically,however, the vector or cassette contains sequences directingtranscription and translation of the relevant gene(s), a selectablemarker and sequences allowing autonomous replication or chromosomalintegration. Suitable vectors comprise a region 5′ of the gene thatcontrols transcriptional initiation and a region 3′ of the DNA fragmentthat controls transcriptional termination. It is most preferred whenboth control regions are derived from genes from the transformed hostcell, although it is to be understood that such control regions need notbe derived from the genes native to the specific species chosen as aproduction host.

Initiation control regions or promoters which are useful to driveexpression of the instant ORF in the desired host cell are numerous andfamiliar to those skilled in the art. Virtually any promoter capable ofdirecting expression of this gene in the selected host cell is suitablefor the present invention. Expression in a host cell can be accomplishedin a transient or stable fashion. Transient expression can beaccomplished by inducing the activity of a regulatable promoter operablylinked to the gene of interest. Stable expression can be achieved by theuse of a constitutive promoter operably linked to the gene of interest.As an example, when the host cell is yeast, transcriptional andtranslational regions functional in yeast cells are provided,particularly from the host species. The transcriptional initiationregulatory regions can be obtained, for example, from: 1.) genes in theglycolytic pathway, such as alcohol dehydrogenase,glyceraldehyde-3-phosphate-dehydrogenase (see U.S. Patent ApplicationNo. 60/482,263), phosphoglycerate mutase (see U.S. Patent ApplicationNo. 60/482,263), fructose-bisphosphate aldolase (see U.S. PatentApplication No. 60/519,971), phosphoglucose-isomerase, phosphoglyceratekinase, etc.; or, 2.) regulatable genes such as acid phosphatase,lactase, metallothionein, glucoamylase, the translation elongationfactor EF1-α (TEF) protein (U.S. Pat. No. 6,265,185), ribosomal proteinS7 (U.S. Pat. No. 6,265,185), etc. Any one of a number of regulatorysequences can be used, depending upon whether constitutive or inducedtranscription is desired, the efficiency of the promoter in expressingthe ORF of interest, the ease of construction and the like.

Nucleotide sequences surrounding the translational initiation codon‘ATG’ have been found to affect expression in yeast cells. If theinstant desaturase is poorly expressed in non-Yarrowia lipolytica yeast,the nucleotide sequences of exogenous genes can be modified to includean efficient yeast translation initiation sequence to obtain optimalgene expression. For expression in yeast, this can be done bysite-directed mutagenesis of an inefficiently expressed gene by fusingit in-frame to an endogenous yeast gene, preferably a highly expressedgene. Alternatively, one can determine the consensus translationinitiation sequence in the host and engineer this sequence intoheterologous genes for their optimal expression in the host of interest.

The termination region can be derived from the 3′ region of the genefrom which the initiation region was obtained or from a different gene.A large number of termination regions are known and functionsatisfactorily in a variety of hosts (when utilized both in the same anddifferent genera and species from where they were derived). Thetermination region usually is selected more as a matter of conveniencerather than because of any particular property. Preferably, thetermination region is derived from a yeast gene, particularlySaccharomyces, Schizosaccharomyces, Candida, Yarrowia or Kluyveromyces.The 3′-regions of mammalian genes encoding γ-interferon and α-2interferon are also known to function in yeast. Termination controlregions may also be derived from various genes native to the preferredhosts. Optionally, a termination site may be unnecessary; however, it ismost preferred if included.

As one of skill in the art is aware, merely inserting a gene into acloning vector does not ensure that it will be successfully expressed atthe level needed. In response to the need for a high expression rate,many specialized expression vectors have been created by manipulating anumber of different genetic elements that control aspects oftranscription, translation, protein stability, oxygen limitation, andsecretion from the host cell. More specifically, some of the molecularfeatures that have been manipulated to control gene expression include:1.) the nature of the relevant transcriptional promoter and terminatorsequences; 2.) the number of copies of the cloned gene and whether thegene is plasmid-borne or integrated into the genome of the host cell;3.) the final cellular location of the synthesized foreign protein; 4.)the efficiency of translation in the host organism; 5.) the intrinsicstability of the cloned gene protein within the host cell; and 6.) thecodon usage within the cloned gene, such that its frequency approachesthe frequency of preferred codon usage of the host cell. Each of thesetypes of modifications are encompassed in the present invention, asmeans to further optimize expression of the Δ12 desaturase describedherein.

Transformation of Microbial Hosts

Once the DNA encoding a polypeptide suitable for expression in anoleaginous yeast has been obtained, it is placed in a plasmid vectorcapable of autonomous replication in a host cell, or it is directlyintegrated into the genome of the host cell. Integration of expressioncassettes can occur randomly within the host genome or can be targetedthrough the use of constructs containing regions of homology with thehost genome sufficient to target recombination within the host locus.Where constructs are targeted to an endogenous locus, all or some of thetranscriptional and translational regulatory regions can be provided bythe endogenous locus.

Where two or more genes are expressed from separate replicating vectors,it is desirable that each vector has a different means of selection andshould lack homology to the other construct(s) to maintain stableexpression and prevent reassortment of elements among constructs.Judicious choice of regulatory regions, selection means and method ofpropagation of the introduced construct(s) can be experimentallydetermined so that all introduced genes are expressed at the necessarylevels to provide for synthesis of the desired products.

Constructs comprising the gene of interest may be introduced into a hostcell by any standard technique. These techniques include transformation(e.g., lithium acetate transformation [Methods in Enzymology,194:186-187 (1991)]), protoplast fusion, biolistic impact,electroporation, microinjection, or any other method that introduces thegene of interest into the host cell. More specific teachings applicablefor oleaginous yeasts (i.e., Yarrowia lipolytica) include U.S. Pat. Nos.4,880,741 and 5,071,764 and Chen, D. C. et al. (Appl MicrobiolBiotechnol. 48(2):232-235 (1997)).

For convenience, a host cell that has been manipulated by any method totake up a DNA sequence (e.g., an expression cassette) will be referredto as “transformed” or “recombinant” herein. The transformed host willhave at least one copy of the expression construct and may have two ormore, depending upon whether the gene is integrated into the genome,amplified, or is present on an extrachromosomal element having multiplecopy numbers. The transformed host cell can be identified by selectionfor a marker contained on the introduced construct. Alternatively, aseparate marker construct may be co-transformed with the desiredconstruct, as many transformation techniques introduce many DNAmolecules into host cells. Typically, transformed hosts are selected fortheir ability to grow on selective media. Selective media mayincorporate an antibiotic or lack a factor necessary for growth of theuntransformed host, such as a nutrient or growth factor. An introducedmarker gene may confer antibiotic resistance, or encode an essentialgrowth factor or enzyme, thereby permitting growth on selective mediawhen expressed in the transformed host. Selection of a transformed hostcan also occur when the expressed marker protein can be detected, eitherdirectly or indirectly. The marker protein may be expressed alone or asa fusion to another protein. The marker protein can be detected by: 1.)its enzymatic activity (e.g., β-galactosidase can convert the substrateX-gal [5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside] to a coloredproduct; luciferase can convert luciferin to a light-emitting product);or 2.) its light-producing or modifying characteristics (e.g., the greenfluorescent protein of Aequorea victoria fluoresces when illuminatedwith blue light). Alternatively, antibodies can be used to detect themarker protein or a molecular tag on, for example, a protein ofinterest. Cells expressing the marker protein or tag can be selected,for example, visually, or by techniques such as FACS or panning usingantibodies. For selection of yeast transformants, any marker thatfunctions in yeast may be used. Desirably, resistance to kanamycin,hygromycin and the amino glycoside G418 are of interest, as well asability to grow on media lacking uracil or leucine.

Following transformation, substrates suitable for the instant Δ12desaturase (and optionally other PUFA enzymes that are co-expressedwithin the host cell) may be produced by the host either naturally ortransgenically, or they may be provided exogenously.

Metabolic Engineering of ω-3 and/or ω-6 Fatty Acid Biosynthesis inMicrobes

Knowledge of the sequence of the present Δ12 desaturase will be usefulfor manipulating ω-3 and/or ω-6 fatty acid biosynthesis in oleaginousyeasts, and particularly, in Yarrowia lipolytica. This may requiremetabolic engineering directly within the PUFA biosynthetic pathway oradditional manipulation of pathways that contribute carbon to the PUFAbiosynthetic pathway. Methods useful for manipulating biochemicalpathways are well known to those skilled in the art.

Techniques to Up-Regulate Desirable Biosynthetic Pathways

Additional copies of desaturase and elongase genes may be introducedinto the host to increase the output of the ω-3 and/or ω-6 fatty acidbiosynthetic pathways, typically through the use of multicopy plasmids.Expression of the desaturase or elongase genes also can be increased atthe transcriptional level through the use of a stronger promoter (eitherregulated or constitutive) to cause increased expression, byremoving/deleting destabilizing sequences from either the mRNA or theencoded protein, or by adding stabilizing sequences to the mRNA (U.S.Pat. No. 4,910,141). Yet another approach to increase expression ofheterologous desaturase or elongase genes is to increase thetranslational efficiency of the encoded mRNAs by replacement of codonsin the native gene with those for optimal gene expression in theselected host microorganism.

Techniques to Down-Regulate Undesirable Biosynthetic Pathways

Conversely, biochemical pathways competing with the ω-3 and/or ω-6 fattyacid-biosynthetic pathways for energy or carbon, or native PUFAbiosynthetic pathway enzymes that interfere with production of aparticular PUFA end-product, may be eliminated by gene disruption ordown-regulated by other means (e.g., antisense mRNA). For genedisruption, a foreign DNA fragment (typically a selectable marker gene)is inserted into the structural gene to be disrupted in order tointerrupt its coding sequence and thereby functionally inactivate thegene. Transformation of the disruption cassette into the host cellresults in replacement of the functional native gene by homologousrecombination with the non-functional disrupted gene (see, for example:Hamilton et al. J. Bacteriol. 171:4617-4622 (1989); Balbas et al. Gene136:211-213 (1993); Gueldener et al. Nucleic Acids Res. 24:2519-2524(1996); and Smith et al. Methods Mol. Cell. Biol. 5:270-277(1996)).

Antisense technology is another method of down-regulating genes when thesequence of the target gene is known. To accomplish this, a nucleic acidsegment from the desired gene is cloned and operably linked to apromoter such that the anti-sense strand of RNA will be transcribed.This construct is then introduced into the host cell and the antisensestrand of RNA is produced. Antisense RNA inhibits gene expression bypreventing the accumulation of mRNA that encodes the protein ofinterest. The person skilled in the art will know that specialconsiderations are associated with the use of antisense technologies inorder to reduce expression of particular genes. For example, the properlevel of expression of antisense genes may require the use of differentchimeric genes utilizing different regulatory elements known to theskilled artisan.

Although targeted gene disruption and antisense technology offereffective means of down-regulating genes where the sequence is known,other less specific methodologies have been developed that are notsequence-based. For example, cells may be exposed to UV radiation andthen screened for the desired phenotype. Mutagenesis with chemicalagents is also effective for generating mutants and commonly usedsubstances include chemicals that affect nonreplicating DNA (e.g., HNO₂and NH₂OH), as well as agents that affect replicating DNA (e.g.,acridine dyes, notable for causing frameshift mutations). Specificmethods for creating mutants using radiation or chemical agents are welldocumented in the art. See, for example: Thomas D. Brock inBiotechnology: A Textbook of Industrial Microbiology, 2^(nd) ed. (1989)Sinauer Associates: Sunderland, Mass.; or Deshpande, Mukund V., Appl.Biochem. Biotechnol., 36:227 (1992).

Another non-specific method of gene disruption is the use oftransposable elements or transposons. Transposons are genetic elementsthat insert randomly into DNA but can be later retrieved on the basis ofsequence to determine where the insertion has occurred. Both in vivo andin vitro transposition methods are known. Both methods involve the useof a transposable element in combination with a transposase enzyme. Whenthe transposable element or transposon is contacted with a nucleic acidfragment in the presence of the transposase, the transposable elementwill randomly insert into the nucleic acid fragment. The technique isuseful for random mutagenesis and for gene isolation, since thedisrupted gene may be identified on the basis of the sequence of thetransposable element. Kits for in vitro transposition are commerciallyavailable [see, for example: 1.) The Primer Island Transposition Kit,available from Perkin Elmer Applied Biosystems, Branchburg, N.J., basedupon the yeast Ty1 element; 2.) The Genome Priming System, availablefrom New-England Biolabs, Beverly, Mass., based upon the bacterialtransposon Tn7; and 3.) the EZ::TN Transposon Insertion Systems,available from Epicentre Technologies, Madison, Wis., based upon the Tn5bacterial transposable element].

Within the context of the present invention, it may be useful tomodulate the expression of the fatty acid biosynthetic pathway by anyone of the methods described above. For example, the present inventionprovides a gene (i.e., a Δ12 desaturase) encoding a key enzyme in thebiosynthetic pathway leading to the production of ω-3 and/or ω-6 fattyacids. It will be particularly useful to express this gene in oleaginousyeasts that produce insufficient amounts of 18:2 fatty acids and tomodulate the expression of this and other PUFA biosynthetic genes tomaximize production of preferred PUFA products using various means formetabolic engineering of the host organism. Likewise, to maximize PUFAproduction with this gene, it may be necessary to disrupt pathways thatcompete for the carbon flux directed toward PUFA biosynthesis. Inalternate embodiments, it may be desirable to disrupt the Δ12 desaturaseherein, to promote synthesis of ω-3 fatty acids while simultaneouslypreventing co-synthesis of ω-6 fatty acids. In another alternateembodiment it will be possible to regulate the production of ω-3/ω-6fatty acids by placing the present Δ12 desaturase gene under the controlof inducible or regulated promoters.

Preferred Microbial Hosts for Recombinant Expression of Δ12 Desaturase

Host cells for expression of the instant gene and nucleic acid fragmentsmay include microbial hosts that grow on a variety of feedstocks,including simple or complex carbohydrates, organic acids and alcohols,and/or hydrocarbons over a wide range of temperature and pH values.Although the genes described in the instant invention have been isolatedfor expression in oleaginous yeast, it is contemplated that becausetranscription, translation and the protein biosynthetic apparatus ishighly conserved, any bacteria, yeast, algae and/or filamentous funguswill be a suitable host for expression of the present nucleic acidfragments.

Preferred microbial hosts are oleaginous organisms, such as oleaginousyeasts. These oleaginous organisms are naturally capable of oilsynthesis and accumulation, wherein the oil can comprise greater thanabout 25% of the cellular dry weight, more preferably greater than about30% of the cellular dry weight, and most preferably greater than about40% of the cellular dry weight. Genera typically identified asoleaginous yeast include, but are not limited to: Yarrowia, Mortierella,Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon andLipomyces. More specifically, illustrative oil-synthesizing yeastsinclude: Rhodosporidium toruloides, Lipomyces starkeyii, L. lipoferus,Candida revkaufi, C. pulcherrima, C. tropicalis, C. utilis, Trichosporonpullans, T. cutaneum, Rhodotorula glutinus, R. graminis, Mortierellaalpina and Yarrowia lipolytica (formerly classified as Candidalipolytica).

Most preferred is the oleaginous yeast Yarrowia lipolytica; and, in afurther embodiment, most preferred are the Y. lipolytica strainsdesignated as ATCC #20362, ATCC #8862, ATCC #18944, ATCC #76982 and/orLGAM S(7)1 (Papanikolaou S., and Aggelis G., Bioresour. Technol.82(1):43-9 (2002)).

Fermentation Processes for PUFA Production

The transformed microbial host cell is grown under conditions thatoptimize activity of fatty acid biosynthetic genes and produce thegreatest and the most economical yield of fatty acids (e.g., LA, whichcan in turn increase the production of various ω-3 and/or ω-6 fattyacids). In general, media conditions that may be optimized include thetype and amount of carbon source, the type and amount of nitrogensource, the carbon-to-nitrogen ratio, the oxygen level, growthtemperature, pH, length of the biomass production phase, length of theoil accumulation phase and the time of cell harvest. Microorganisms ofinterest, such as oleaginous yeast, are grown in complex media (e.g.,yeast extract-peptone-dextrose broth (YPD)) or a defined minimal mediathat lacks a component necessary for growth and thereby forces selectionof the desired expression cassettes (e.g., Yeast Nitrogen Base (DIFCOLaboratories, Detroit, Mich.)).

Fermentation media in the present invention must contain a suitablecarbon source. Suitable carbon sources may include, but are not limitedto: monosaccharides (e.g., glucose, fructose), disaccharides (e.g.,lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch,cellulose or mixtures thereof), sugar alcohols (e.g., glycerol) ormixtures from renewable feedstocks (e.g., cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt). Additionally,carbon sources may include alkanes, fatty acids, esters of fatty acids,monoglycerides, diglycerides, triglycerides, phospholipids and variouscommercial sources of fatty acids including vegetable oils (e.g.,soybean oil) and animal fats. Additionally, the carbon substrate mayinclude one-carbon substrates (e.g., carbon dioxide or methanol) forwhich metabolic conversion into key biochemical intermediates has beendemonstrated. Hence it is contemplated that the source of carbonutilized in the present invention may encompass a wide variety ofcarbon-containing substrates and will only be limited by the choice ofthe host organism. Although all of the above mentioned carbon substratesand mixtures thereof are expected to be suitable in the presentinvention, preferred carbon substrates are sugars and/or fatty acids.Most preferred is glucose and/or fatty acids containing between 10-22carbons.

Nitrogen may be supplied from an inorganic (e.g., (NH₄)₂SO₄) or organicsource (e.g., urea or glutamate). In addition to appropriate carbon andnitrogen sources, the fermentation media must also contain suitableminerals, salts, cofactors, buffers, vitamins, and other componentsknown to those skilled in the art suitable for the growth of themicroorganism and promotion of the enzymatic pathways necessary for PUFAproduction. Particular attention is given to several metal ions (e.g.,Mn⁺², Co⁺², Zn⁺², Mg⁺²) that promote synthesis of lipids and PUFAs(Nakahara, T. et al., Ind. Appl. Single Cell Oils, D. J. Kyle and R.Colin, eds. pp 61-97 (1992)).

Preferred growth media in the present invention are common commerciallyprepared media, such as Yeast Nitrogen Base (DIFCO Laboratories,Detroit, Mich.). Other defined or synthetic growth media may also beused and the appropriate medium for growth of the particularmicroorganism will be known by one skilled in the art of microbiology orfermentation science. A suitable pH range for the fermentation istypically between about pH 4.0 to pH 8.0, wherein pH 5.5 to pH 7.0 ispreferred as the range for the initial growth conditions. Thefermentation may be conducted under aerobic or anaerobic conditions,wherein microaerobic conditions are preferred.

Typically, accumulation of high levels of PUFAs in oleaginous yeastcells requires a two-stage process, since the metabolic state must be“balanced” between growth and synthesis/storage of fats. Thus, mostpreferably, a two-stage fermentation process is necessary for theproduction of PUFAs in oleaginous yeast. In this approach, the firststage of the fermentation is dedicated to the generation andaccumulation of cell mass and is characterized by rapid cell growth andcell division. In the second stage of the fermentation, it is preferableto establish conditions of nitrogen deprivation in the culture topromote high levels of lipid accumulation. The effect of this nitrogendeprivation is to reduce the effective concentration of AMP in thecells, thereby reducing the activity of the NAD-dependent isocitratedehydrogenase of mitochondria. When this occurs, citric acid willaccumulate, thus forming abundant pools of acetyl-CoA in the cytoplasmand priming fatty acid synthesis. Thus, this phase is characterized bythe cessation of cell division followed by the synthesis of fatty acidsand accumulation of oil.

Although cells are typically grown at about 30° C., some studies haveshown increased synthesis of unsaturated fatty acids at lowertemperatures (Yongmanitchai and Ward, Appl. Environ. Microbiol.57:419-25 (1991)). Based on process economics, this temperature shiftshould likely occur after the first phase of the two-stage fermentation,when the bulk of the organisms' growth has occurred.

It is contemplated that a variety of fermentation process designs may beapplied, where commercial production of omega fatty acids using theinstant Δ12 desaturase is desired. For example, commercial production ofPUFAs from a recombinant microbial host may be produced by a batch,fed-batch or continuous fermentation process.

A batch fermentation process is a closed system wherein themedia-composition is set at the beginning of the process and not subjectto further additions beyond those required for maintenance of pH andoxygen level during the process. Thus, at the beginning of the culturingprocess the media is inoculated with the desired organism and growth ormetabolic activity is permitted to occur without adding additionalsubstrates (i.e., carbon and nitrogen sources) to the medium. In batchprocesses the metabolite and biomass compositions of the system changeconstantly up to the time the culture is terminated. In a typical batchprocess, cells proceed through a static lag phase to a high-growth logphase and finally to a stationary phase, wherein the growth rate isdiminished or halted. Left untreated, cells in the stationary phase willeventually die. A variation of the standard batch process is thefed-batch process, wherein the substrate is continually added to thefermentor over the course of the fermentation process. A fed-batchprocess is also suitable in the present invention. Fed-batch processesare useful when catabolite repression is apt to inhibit the metabolismof the cells or where it is desirable to have limited amounts ofsubstrate in the media at any one time. Measurement of the substrateconcentration in fed-batch systems is difficult and therefore may beestimated on the basis of the changes of measurable factors such as pH,dissolved oxygen and the partial pressure of waste gases (e.g., CO₂).Batch and fed-batch culturing methods are common and well known in theart and examples may be found in Thomas D. Brock in Biotechnology: ATextbook of Industrial Microbiology, 2^(nd) ed., (1989) SinauerAssociates: Sunderland, Mass.; or Deshpande, Mukund V., Appl. Biochem.Biotechnol., 36:227 (1992), herein incorporated by reference.

Commercial production of omega fatty acids using the instant Δ12desaturase may also be accomplished by a continuous fermentation processwherein a defined media is continuously added to a bioreactor while anequal amount of culture volume is removed simultaneously for productrecovery. Continuous cultures generally maintain the cells in the logphase of growth at a constant cell density. Continuous orsemi-continuous culture methods permit the modulation of one factor orany number of factors that affect cell growth or end productconcentration. For example, one approach may limit the carbon source andallow all other parameters to moderate metabolism. In other systems, anumber of factors affecting growth may be altered continuously while thecell concentration, measured by media turbidity, is kept constant.Continuous systems strive to maintain steady state growth and thus thecell growth rate must be balanced against cell loss due to media beingdrawn off the culture. Methods of modulating nutrients and growthfactors for continuous culture processes, as well as techniques formaximizing the rate of product formation, are well known in the art ofindustrial microbiology and a variety of methods are detailed by Brock,supra.

Purification of PUFAs

The PUFAs may be found in the host microorganism as free fatty acids orin esterified forms such as acylglycerols, phospholipids, sulfolipids orglycolipids, and may be extracted from the host cell through a varietyof means well-known in the art. One review of extraction techniques,quality analysis and acceptability standards for yeast lipids is that ofZ. Jacobs (Critical Reviews in Biotechnology 12(5/6):463-491 (1992)). Abrief review of downstream processing is also available by A. Singh andO. Ward (Adv. Appl. Microbiol. 45:271-312 (1997)).

In general, means for the purification of PUFAs may include extractionwith organic solvents, sonication, supercritical fluid extraction (e.g.,using carbon dioxide), saponification and physical means such aspresses, or combinations thereof. Of particular interest is extractionwith methanol and chloroform in the presence of water (E. G. Bligh & W.J. Dyer, Can. J. Biochem. Physiol. 37:911-917 (1959)). Where desirable,the aqueous layer can be acidified to protonate negatively-chargedmoieties and thereby increase partitioning of desired products into theorganic layer. After extraction, the organic solvents can be removed byevaporation under a stream of nitrogen. When isolated in conjugatedforms, the products may be enzymatically or chemically cleaved torelease the free fatty acid or a less complex conjugate of interest, andcan then be subject to further manipulations to produce a desired endproduct. Desirably, conjugated forms of fatty acids are cleaved withpotassium hydroxide.

If further purification is necessary, standard methods can be employed.Such methods may include extraction, treatment with urea, fractionalcrystallization, HPLC, fractional distillation, silica gelchromatography, high-speed centrifugation or distillation, orcombinations of these techniques. Protection of reactive groups, such asthe acid or alkenyl groups, may be done at any step through knowntechniques (e.g., alkylation, iodination). Methods used includemethylation of the fatty acids to produce methyl esters. Similarly,protecting groups may be removed at any step. Desirably, purification offractions containing GLA, STA, ARA, DHA and EPA may be accomplished bytreatment with urea and/or fractional distillation.

DESCRIPTION OF PREFERRED EMBODIMENTS

The ultimate goal of the work described herein is the development of anoleaginous yeast that accumulates oils enriched in ω-3 and/or ω-6 PUFAs.Toward this end, Δ12 desaturases must be identified that functionefficiently in oleaginous yeasts, to enable synthesis and highaccumulation of preferred PUFAs in these hosts. Identification ofefficient Δ12 desaturases is also necessary to manipulate the ratio ofω-3 to ω-6 PUFAs produced in host cells.

In the present invention, Applicants have isolated and cloned the onlygene in Yarrowia lipolytica that encodes a Δ12 desaturase enzyme.Confirmation of this gene's activity was provided based upon: 1.) thelack of detectable LA in a strain wherein disruption of the native Δ12desaturase by targeted gene replacement through homologous recombinationhad occurred (Example 2); 2.) restoration of LA biosynthesis(complementation) in the disrupted strain upon transformation with thechimeric gene (Example 4); and 3.) the overproduction of LA in wild typecells upon transformation with the chimeric gene (Example 4). Thus, thisΔ12 desaturase gene is useful for expression in various microbial hosts,and particularly for overexpression in oleaginous yeasts (e.g., thenative host Yarrowia lipolytica). Additional benefits may result sinceexpression of the Δ12 desaturase can also be put under the control ofstrong constitutive or regulated promoters that do not have theregulatory constraints of the native gene.

Following the initial demonstration of functionality of the Δ12desaturase in Yarrowia lipolytica, the Applicants then explored methodsof optimizing PUFA production within this model host organism.Specifically, a Δ12 desaturase-disrupted host strain of Y. lipolyticawas created and transformed with an expression cassette comprising aheterologous Δ6 desaturase, elongase, Δ5 desaturase and Δ17 desaturase.When fed ALA as a substrate, the transformed host was able to produceSTA without co-synthesis of any ω-6 fatty acid (Example 8). Thus, thiswork demonstrated that upon transformation with appropriate genes of theω-3 biosynthetic pathway and feeding of ALA as a substrate, only ω-3fatty acids (e.g., ETA, EPA, DPA, DHA) could be synthesized (i.e.,without co-synthesis of ω-6 fatty acids) in Yarrowia strains lacking Δ12desaturase activity.

EXAMPLES

The present invention is further defined in the following Examples. Itshould be understood that these Examples, while indicating preferredembodiments of the invention, are given by way of illustration only.From the above discussion and these Examples, one skilled in the art canascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

General Methods

Standard recombinant DNA and molecular cloning techniques used in theExamples are well known in the art and are described by: 1.) Sambrook,J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A LaboratoryManual; Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y. (1989)(Maniatis); 2.) T. J. Silhavy, M. L. Bennan, and L. W. Enquist,Experiments with Gene Fusions; Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y. (1984); and 3.) Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, published by Greene Publishing Assoc.and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth ofmicrobial cultures are well known in the art. Techniques suitable foruse in the following Examples may be found as set out in Manual ofMethods for General Bacteriology (Phillipp Gerhardt, R. G. E. Murray,Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg andG. Briggs Phillips, Eds), American Society for Microbiology: Washington,D.C. (1994)); or by Thomas D. Brock in Biotechnology: A Textbook ofIndustrial Microbiology, 2^(nd) ed., Sinauer Associates: Sunderland,Mass. (1989). All reagents, restriction enzymes and materials used forthe growth and maintenance of microbial cells were obtained from AldrichChemicals (Milwaukee, Wis.), DIFCO Laboratories (Detroit, Mich.),GIBCO/BRL (Gaithersburg, Md.), or Sigma Chemical Company (St. Louis,Mo.), unless otherwise specified.

E. coli TOP10 cells and E. coli Electromax DH10B cells were obtainedfrom Invitrogen (Carlsbad, Calif.). Max Efficiency competent cells of E.coli DH5α were obtained from GIBCO/BRL (Gaithersburg, Md.). E. coli(XL1-Blue) competent cells were purchased from the Stratagene Company(San Diego, Calif.). E. coli strains were typically grown at 37° C. onLuria Bertani (LB) plates.

General molecular cloning was performed according to standard methods(Sambrook et al., supra). Oligonucleotides were synthesized bySigma-Genosys (Spring, Tex.). PCR products were cloned into Promega'spGEM-T-easy vector (Madison, Wis.).

DNA sequence was generated on an ABI Automatic sequencer using dyeterminator technology (U.S. Pat. No. 5,366,860; EP 272,007) using acombination of vector and insert-specific primers. Sequence editing wasperformed in Sequencher (Gene Codes Corporation, Ann Arbor, Mich.). Allsequences represent coverage at least two times in both directions.Comparisons of genetic sequences were accomplished using DNASTARsoftware (DNA Star, Inc.).

The meaning of abbreviations is as follows: “sec” means second(s), “min”means minute(s), “h” means hour(s), “d” means day(s), “μL” meansmicroliter(s), “mL” means milliliter(s), “L” means liter(s), “μM” meansmicromolar, “mM” means millimolar, “M” means molar, “mmol” meansmillimole(s), “μmole” mean micromole(s), “g” means gram(s), “μg” meansmicrogram(s), “ng” means nanogram(s), “U” means unit(s), “bp” means basepair(s) and “kB” means kilobase(s).

Cultivation of Yarrowia lipolytica

Yarrowia lipolytica strains ATCC #76982 and ATCC #90812 were purchasedfrom the American Type Culture Collection (Rockville, Md.). Y.lipolytica strains were usually grown at 28° C. on YPD agar (1% yeastextract, 2% bactopeptone, 2% glucose, 2% agar). For selection oftransformants, minimal medium (0.17% yeast nitrogen base (DIFCOLaboratories, Detroit, Mich.) without ammonium sulfate or amino acids,2% glucose, 0.1% proline, pH 6.1) was used. Supplements of adenine,leucine, lysine and/or uracil were added as appropriate to a finalconcentration of 0.01%.

Fatty Acid Analysis of Yarrowia lipolytica

For fatty acid analysis, cells were collected by centrifugation andlipids were extracted as described in Bligh, E. G. & Dyer, W. J. (Can.J. Biochem. Physiol. 37:911-917 (1959)). Fatty acid methyl esters wereprepared by transesterification of the lipid extract with sodiummethoxide (Roughan, G., and Nishida I. Arch Biochem Biophys.276(1):38-46 (1990)) and subsequently analyzed with a Hewlett-Packard6890 GC fitted with a 30-m×0.25 mm (i.d.) HP-INNOWAX (Hewlett-Packard)column. The oven temperature was from 170° C. (25 min hold) to 185° C.at 3.5° C./min.

For direct base transesterification, Yarrowia culture (3 mL) washarvested, washed once in distilled water, and dried under vacuum in aSpeed-Vac for 5-10 min. Sodium methoxide (100 μl of 1%) was added to thesample, and then the sample was vortexed and rocked for 20 min. Afteradding 3 drops of 1 M NaCl and 400 μl hexane, the sample was vortexedand spun. The upper layer was removed and analyzed by GC as describedabove.

Example 1 Construction of Plasmids Suitable for Gene Expression inYarrowia lipolytica

The present Example describes the construction of plasmids pY5, pY5-4,pY5-13 and pY5-20.

Construction of Plasmid pY5

The plasmid pY5, a derivative of pINA532 (a gift from Dr. ClaudeGaillardin, Insitut National Agronomics, Centre de biotechnologieAgro-Industrielle, laboratoire de Genetique Moleculaire et CellularieINRA-CNRS, F-78850 Thiverval-Grignon, France), was constructed forexpression of heterologous genes in Yarrowia lipolytica, as diagrammedin FIG. 3.

First, the partially-digested 3598 bp EcoRI fragment containing theARS18 sequence and LEU2 gene of pINA532 was subcloned into the EcoRIsite of pBluescript (Strategene, San Diego, Calif.) to generate pY2. TheTEF promoter (Muller S., et al. Yeast, 14: 1267-1283 (1998)) wasamplified from Yarrowia lipolytica genomic DNA by PCR using TEF5′ (SEQID NO:1) and TEF3′ (SEQ ID NO:2) as primers. PCR amplification wascarried out in a 50 μl total volume containing: 100 ng Yarrowia genomicDNA, PCR buffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl(pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (finalconcentration), 200 μM each deoxyribonucleotide triphosphate, 10 pmoleof each primer and 1 μl of PfuTurbo DNA polymerase (Stratagene).Amplification was carried out as follows: initial denaturation at 95° C.for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56°C. for 30 sec, 72° C. for 1 min. A final extension cycle of 72° C. for10 min was carried out, followed by reaction termination at 4° C. The418 bp PCR product was ligated into pCR-Blunt to generate pIP-tef. TheBamHI/EcoRV fragment of pIP-tef was subcloned into the BamHI/SmaI sitesof pY2 to generate pY4.

The XPR2 transcriptional terminator was amplified by PCR using pINA532as template and XPR5′ (SEQ ID NO:3) and XPR3′ (SEQ ID NO:4) as primers.The PCR amplification was carried out in a 50 μl total volume, using thecomponents and conditions described above. The 179 bp PCR product wasdigested with SacII and then ligated into the SacII site of pY4 togenerate pY5. Thus, pY5 (shown in FIGS. 3 and 4) is useful as aYarrowia-E. coli shuttle plasmid containing:

-   -   1.) a Yarrowia autonomous replication sequence (ARS18);    -   2.) a ColE1 plasmid origin of replication;    -   3.) an ampicillin-resistance gene (Amp^(R)), for selection in E.        coli;    -   4.) a Yarrowia LEU2 gene (E.C. 1.1.1.85, encoding        isopropylmalate isomerase), for selection in Yarrowia;    -   5.) the translation elongation promoter (TEF P), for expression        of heterologous genes in Yarrowia; and    -   6.) the extracellular protease gene terminator (XPR2) for        transcriptional termination of heterologous gene expression in        Yarrowia.        Construction of Plasmids pY-4, pY5-13 And pY5-20

pY5-4 and pY5-13 (FIG. 4) were constructed as derivatives of pY5 tofaciliate subcloning and heterologous gene expression in Yarrowialipolytica.

Specifically, pY5-4 was constructed by three rounds of site-directedmutagenesis using pY5 as template. A NcoI site located inside the Leu2reporter gene was eliminated from pY5 using oligonucleotides YL1 and YL2(SEQ ID NOs:5 and 6) to generate pY5-1. A NcoI site was introduced intopY5-1 between the TEF promoter and XPR2 transcriptional terminator bysite-directed mutagenesis using oligonucleotides YL3 and YL4 (SEQ IDNOs:7 and 8) to generate pY5-2. A PacI site was then introduced intopY5-2 between the TEF promoter and XPR2 transcriptional terminator usingoligonucleotides YL23 and YL24 (SEQ ID NOs:9 and 10) to generate pY5-4.

pY5-13 was constructed by 6 rounds of site-directed mutagenesis usingpY5 as template. Both SalI and ClaI sites were eliminated from pY5 bysite-directed mutagenesis using oligonucleotides YL5 and YL6 (SEQ IDNOs:11 and 12) to generate pY5-5. A SalI site was introduced into pY5-5between the Leu2 gene and the TEF promoter by site-directed mutagenesisusing oligonucleotides YL9 and YL10 (SEQ ID NOs:13 and 14) to generatepY5-6. A PacI site was introduced into pY5-6 between the LEU2 gene andARS18 using oligonucleotides YL7 and YL8 (SEQ ID NOs:15 and 16) togenerate pY5-8. A NcoI site was introduced into pY5-8 around thetranslation start codon of the TEF promoter using oligonucleotides YL3and YL4 (SEQ ID NOs:7 and 8) to generate pY5-9. The NcoI site inside theLeu2 gene of pY5-9 was eliminated using YL1 and YL2 oligonucleotides(SEQ ID NOs:5 and 6) to generate pY5-12. Finally, a BsiWI site wasintroduced into pY5-12 between the ColEI and XPR2 region usingoligonucleotides YL61 and YL62 (SEQ ID NOs:17 and 18) to generatepY5-13.

Plasmid pY20 is a derivative of pY5. It was constructed by inserting aNot I fragment containing a chimeric hygromycin resistance gene(hygromycin-B phosphotransferase; GenBank Accession No. P00557) into theNot/site of pY5. The chimeric gene had the hygromycin resistance ORFunder the control of a Yarrowia lipolytica TEF promoter.

Example 2 Cloning of the Partial Yarrowia lipolytica Δ12 Desaturase andDisruption of the Endogenous Δ12 Desaturase Gene

Based on the fatty acid composition of wildtype Yarrowia lipolytica(ATCC #76982) which demonstrated that the organism could make LA (18:2)but not ALA (18:3), it was assumed that Y. lipolytica would likelycontain gene(s) having Δ12 desaturase activity but not Δ15 desaturaseactivity. Thus, the present Example describes the use of degenerate PCRprimers to isolate a partial coding sequence of the Y. lipolytica Δ12desaturase and the use of the partial sequence to disrupt the nativegene.

Cloning of the Partial Putative Δ12 Desaturase Sequence from Y.lipolytica by PCR Using Degenerate PCR Primers

Genomic DNA was isolated from Y. lipolytica (ATCC #76982) using DNeasyTissue Kit (Qiagen, Catalog #69504) and resuspended in kit buffer AE ata DNA concentration of 0.5 μg/μl. PCR amplifications were performedusing the genomic DNA as template and several sets of degenerate primersmade to amino acid sequences conserved between different fungal Δ12desaturases (i.e., Mortierella alpina, Mucor rouxii, Emericella nidulansand Pichia augusta). The best results were obtained with a set of upperand lower degenerate primers, P73 and P76, respectively, as shown in theTable below.

TABLE 3 Degenerate Primers Used For Amplification Of The PartialPutative Δ12 Desaturase Degenerate Corresponding Primer Nucleotide AminoAcid Set Description Sequence Sequence P73 (32) 26- 5′- WVLGHECGH mersTGGGTCCTGGGCCAYG (SEQ ID NO:20) ARTGYGGNCA-3′ (SEQ ID NO:19) P76 (64)30- 5′- (M/I)PFYHAEEAT mers GGTGGCCTCCTCGGCG (SEQ ID NO:22)TGRTARAANGGNAT- 3′ (SEQ ID NO:21) [Note: Abbreviations are standard fornudeotides and proteins. The-nucleic acid degeneracy code used is asfollows: R = A/G; Y = C/T; and N = A/C/G/T.]

The PCR was carried out in an Eppendorf Mastercycler Gradientthermocycler according to the manufacturer's recommendations.Amplification was carried out as follows: initial denaturation at 95° C.for 1 min, followed by 30 cycles of denaturation at 95° C. for 30 sec,annealing at 58° C. for 1 min, and elongation at 72° C. for 1 min. Afinal elongation cycle at 72° C. for 10 min was carried out, followed byreaction termination at 4° C.

The expected (ca. 740 bp) size PCR product was detected by agarose gelelectrophoresis, isolated, purified, cloned into a pTA vector(Invitrogen) and sequenced. The resultant sequence (contained within SEQID NO:23) had homology to known Δ12 desaturases, based on BLAST programanalysis (Basic Local Alignment Search Tool; Altschul, S. F., et al., J.Mol. Biol. 215:403-410 (1993).

Targeted Disruption of the Yarrowia lipolytica Δ12 Desaturase Gene

Targeted disruption of the native Δ12 desaturase gene in Y. lipolytica#76982 was carried out by homologous recombination-mediated replacementof the Δ12 desaturase gene with a targeting cassette designated aspY23D12. pY23D12 was derived from plasmid pY20 (Example 1).Specifically, pY23D12 was created by inserting a 642 bp Hind III/Eco RIfragment into similarly linearized pY20. This 642 bp fragment consistedof (in 5′ to 3′ orientation): 3′ homologous sequence from position +718to +1031 (of the coding sequence (ORF) in SEQ ID NO:23), a Bgl IIrestriction site and 5′ homologous sequence from position +403 to +717(of the coding sequence (ORF) in SEQ ID NO:23). The fragment wasprepared by PCR amplification of 3′ and 5′ sequences from the 642 bp PCRproduct using sets of PCR primers P99 and P100 (SEQ ID NOs:25 and 26)and P101 and P102 (SEQ ID NOs:27 and 28), respectively.

pY23D12 was linearized by Bgl II restriction digestion and transformedinto mid-log phase Y. lipolytica cells by the lithium acetate methodaccording to the method of Chen, D. C. et al. (Appl MicrobiolBiotechnol. 48(2):232-235 (1997)). Briefly, Y. lipolytica ATCC #76982was streaked onto a YPD plate and grown at 30° C. for approximately 18hr. Several large loopfuls of cells were scraped from the plate andresuspended in 1 mL of transformation buffer containing:

-   -   2.25 mL of 50% PEG, average MW 3350;    -   0.125 mL of 2 M Li acetate, pH 6.0;    -   0.125 mL of 2 M DTT; and,    -   50 μg sheared salmon sperm DNA.

About 500 ng of plasmid DNA were incubated in 100 μl of resuspendedcells, and maintained at 39° C. for 1 hr with vortex mixing at 15 minintervals. The cells were plated onto YPD hygromycin selection platesand maintained at 30° C. for 2 to 3 days.

Four hygromycin-resistant colonies were isolated and screened fortargeted disruption by PCR. One set of PCR primers (P119 [SEQ ID NO:29]and P120 [SEQ ID NO:30]) was designed to amplify a specific junctionfragment following homologous recombination. Another set of PCR primers(P121 [SEQ ID NO:31] and P122 [SEQ ID NO:32]) was designed to detect thenative gene. Three of the four hygromycin-resistant colonies werepositive for the junction fragment and negative for the native fragment,thus confirming targeted integration.

Determination of Fatty Acid Profile in the Δ12 Desaturase-DisruptedStrain

Disruption of the Δ12 desaturase gene was further confirmed by GCanalysis of the total lipids in one of the disrupted strains, designatedas “Q-d12D”. Single colonies of wild type (ATCC #76982) and Q-d12D Y.lipolytica were each grown in 3 mL minimal media (formulation/L: 20 gglucose, 1.7 g yeast nitrogen base, 1 g L-proline, 0.1 g L-adenine, 0.1g L-lysine, pH 6.1) at 30° C. to an OD₆₀₀˜1.0. The cells were harvested,washed in distilled water, speed vacuum dried and subjected to directtrans-esterification and GC analysis (as described in the GeneralMethods).

The fatty acid profile of wildtype Yarrowia and the transformant Q-d12Dcomprising the disrupted Δ12 desaturase are shown below in Table 4.Fatty acids are identified as 16:0 (palmitate), 16:1 (palmitoleic acid),18:0, 18:1 (oleic acid) and 18:2 (LA); and the composition of each ispresented as a % of the total fatty acids.

TABLE 4 Fatty Acid Composition (% Of Total Fatty Acids) In Wildtype AndTransformant Yarrowia lipolytica Strain 16:0 16:1 18:0 18:1 18:2 Wildtype 11 14 2 33 34 Q-d12D disrupted 6 15 1 74 nd *nd = not detectableResults indicated that the native Δ12 desaturase gene in the Q-d12Dstrain was inactivated. Thus, it was possible to conclude that there wasonly one gene encoding a functional Δ12 desaturase in Yarrowialipolytica ATCC #76982.

Example 3 Cloning of the Full-Length Yarrowia lipolytica Δ12 DesaturaseGene

The present Example describes the recovery of the genomic sequencesflanking the disrupted gene by plasmid rescue, using the sequence in therescued plasmid to PCR the intact open reading frame of the native gene.The full-length gene and its deduced amino acid sequence is compared toother fungal desaturases.

Plasmid Rescue of the Yarrowia lipolytica Δ12 Desaturase Gene

Since the Δ12 desaturase gene was disrupted by the insertion of theentire pY23D12 vector that also contained an E. coliampicillin-resistant gene and E. coli ori, it was possible to rescue theflanking sequences in E. coli. For this, genomic DNA of Y. lipolyticastrain Q-d12D (carrying the disrupted Δ12 desaturase gene; Example 2)was isolated using the DNeasy Tissue Kit. Then, 10 μg of the genomic DNAwas digested with 50 μl of restriction enzymes Age I, Avr II, Nhe I andSph I in a reaction volume of 200 μl. Digested DNA was extracted withphenol:chloroform and resuspended in 40 μl deionized water. The digestedDNA (10 μl) was self-ligated in a 200 μl ligation mixture containing 3 UT4 DNA ligase. Ligation was carried out at 16° C. for 12 hrs. Theligated DNA was extracted with phenol:chloroform and resuspended in 40μl deionized water. Finally, 1 μl of the resuspended ligated DNA wasused to transform E. coli by electroporation and plated onto LB platescontaining ampicillin (Ap). Ap-resistant colonies were isolated andanalyzed for the presence of plasmids by miniprep. The following insertsizes were found in the recovered or rescued plasmids (Table 5):

TABLE 5 Insert Sizes Of Recovered Plasmids, According To RestrictionEnzyme Enzyme Plasmid Insert Size (kB) AgeI 1.6 AvrII 2.5 NheI 9.4 SphI6.6Sequencing of the plasmids was initiated with sequencing primers P99(SEQ ID NO:25) and P102 (SEQ ID NO:28).

Based on the sequencing results, a full-length gene encoding theYarrowia lipolytica Δ12 desaturase gene was assembled (1-936 bp; SEQ IDNO:23). Specifically, SEQ ID NO:23 encoded an open reading frame of 1257bases (nucleotides +283 to +1539), while the deduced amino acid sequencewas 419 residues in length (SEQ ID NO:24).

The Yarrowia lipolytica Δ12 desaturase protein (SEQ ID NO:24) was usedas a query against available sequence databases of filamentous fungi,including: 1.) public databases of Neurospora crassa, Magnaporthegrisea, Aspergillus nidulans and Kluyveromuces lactis; and 2.) a DuPontEST library of Fusarium moniliforme strain M-8114 (E.I. du Pont deNemours and Co., Inc., Wilmington, Del.) (F. moniliforme strain M-8114available from the Fusarium Research Center, University Park, Pa.; seealso Plant Disease 81(2): 211-216. (1997)). These BLAST searchesidentified the following homologs (Table 6).

TABLE 6 Description of Δ12 Desaturase Homologs Source Symbol OrganismContig 1.122 (scaffold 9) in the A. nidulans An1 Aspergillus genomeproject (sponsored by the Center nidulans for Genome Research (CGR),Cambridge, MA. Contig 1.15 (scaffold 1) in the A. nidulans An2Aspergillus genome project; AAG36933 nidulans DuPont EST sequencedatabase, U.S. Fm1 Fusarium Provisional Application No. 60/519191moniliforme DuPont EST sequence database, U.S. Fm2 Fusarium ProvisionalApplication No. 60/519191 moniliforme Ctg4369-0000002-2.1 in theGenolevures Kl Kluyveromyces project. lactis Locus MG08474.1 in contig2.1597 in the M. Mg1 Magnaporthe grisea genome project (sponsored by thegrisea CGR and International Rice Blast Genome Consortium. LocusMG01985.1 in contig 2.375 in the M. Mg2 Magnaporthe grisea genomeproject grisea GenBank Accession No. AABX01000374 Nc1 Neurospora crassaGenBank Accession No. AABX01000577 Nc2 Neurospora crassaAll of the homologs were either unannotated or annotated as a fatty aciddesaturase. Furthermore, the nucleotide sequences from A. nidulans wereincomplete and/or genomic with putative intron sequences; the Applicantsmade a tentative assembly of the deduced amino acids for comparison withamino acid sequences from the other homologs.

A comparison of the deduced amino acid sequence of the Yarrowialipolytica Δ12 desaturase (SEQ ID NO:24) was made with the fungalhomologs shown above in Table 6 and other known Δ12 desaturases, asdescribed below in Table 7.

TABLE 7 Known Δ12 Desaturases Source Symbol Organism GenBank AccessionNo. AAG36933 En Emericella nidulans GenBank Accession No. AF110509 MaMortierella alpina GenBank Accession No. AB020033 MaB Mortierella alpinaGenBank Accession No. AAL13300; MaC Mortierella alpina AF417244 GenBankAccession No. AF161219 Mr Mucor rouxii Ctg1334-0000001-1.1. Pa Pichiaaugusta (see Genolevures project.)

Specifically, the analysis was performed using the ClustalW alignmentalgorithm (Slow/Accurate, Gonnet option; Thompson et. al., Nucleic AcidsRes. 22:4673-4680 (1994)) of the DNASTAR software package (DNASTAR Inc.,Madison, Wis.). This comparison revealed the Pair Distances shown inFIG. 5, wherein “Yl” corresponds to the Yarrowia lipolytica Δ12desaturase. Percent similarity and divergence are shown in the upper andlower triangles, respectively. Thus, the Y. lipolytica Δ12 desaturasewas at least 53% identical to the other Δ12 desaturase homologs (havingmaximal identity to the A. nidulans sequence (An2)).

Example 4 Expression of Yarrowia lipolytica Δ12 Desaturase ORF Under theControl of a Heterologous Yarrowia Promoter

The present Example describes the expression of the Δ12 desaturase ORFin a chimeric gene under the control of a heterologous (non-Δ12desaturase) Yarrowia promoter to complement the Δ12 desaturase-disruptedmutant and enable the overproduction of LA in the wildtype strain.

Expression of Y. lipolytica Δ12 Desaturase in Yarrowia lipolytica.

The ORF encoding the Y. lipolytica Δ12 desaturase was PCR amplifiedusing upper primer P147 (SEQ ID NO:33) and lower primer P148 (SEQ IDNO:34) from the genomic DNA of Y. lipolytica ATCC #76982. The correctsized (1260 bp) fragment was isolated, purified, digested with Nco I andNot I and cloned into NcoI-Not I cut pY5-13 vector (Example 1), suchthat the gene was under the control of the TEF promoter. Correcttransformants were confirmed by miniprep analysis and the resultantplasmid was designated pY25-d12d.

Plasmids pY5-13 (the “control”) and pY25-d12d were each individuallytransformed into Y. lipolytica ATCC #76982 wild-type (WT) andd12d-disrupted strains (Q-d12D, also referred to as “d12KO” in the Tablebelow) and selected on Bio101 DOB/CSM-Leu plates.

Single colonies of transformants were grown up and GC analyzed asdescribed in the General Methods. Results are shown in the Table below.Fatty acids are identified as 16:0, 16:1, 18:0, 18:1 (oleic acid) and18:2 (LA); and the composition of each is presented as a % of the totalfatty acids. “D12d SC” was calculated according to the followingformula: ([18:2]/[18:1+18:2])*100 and represents percent substrateconversion.

TABLE 8 Fatty Acid Composition (% Of Total Fatty Acids) % % % % % D12dStrain Plasmid 16:0 16:1 18:0 18:1 18:2 SC D12KO pY5-13 8 10 2 80 nd 0D12KO pY25-d12d 11 8 2 34 45 57 WT pY5-13 10 10 1 32 47 59 WT pY25-d12d12 7 2 21 59 74 *nd = not detectable

The results showed that the Δ12 desaturase promoter was equivalent instrength to the TEF promoter (57% substrate conversion in the d12KOstrain expressing the Δ12 desaturase under the control of the TEFpromoter, compared to 59% substrate conversion in the wild type strainexpressing the Δ12 desaturase under the control of the native Δ12desaturase promoter). On this basis, it is expected that the Δ12desaturase promoter can be used for heterologous expression of otherORFs in Yarrowia.

Additionally, the results demonstrated that overexpression of the Δ12desaturase in wild type cells resulted in even higher levels of LAproduction (18:2). Specifically, 74% substrate conversion was observedin the wildtype strain overexpressing the Δ12 desaturase under thecontrol of the TEF promoter, as opposed to only 59% substrate conversionin the wild type strain. On the basis of these results, it would beexpected that overexpression of the Δ12 desaturase, in combination ofother genes for PUFA biosynthesis (e.g., a Δ6 desaturase, elongase, Δ5desaturase, Δ17 desaturase), would result in higher production of ω-3and/or ω-6 PUFAs. Additionally, it would be expected that disruption ofthe native Δ12 desaturase and expression of other genes for PUFAbiosynthesis (e.g., a Δ6 desaturase, elongase, Δ5 desaturase, Δ17desaturase) would result in production of “pure” ω-3 PUFAs, withoutco-synthesis of any ω-6 PUFAs.

Example 5 Selection of Δ6 Desaturase, Δ5 Desaturase, Δ17 Desaturase andHigh Affinity PUFA Elongase Genes For Expression in Yarrowia lipolytica

Prior to the introduction of specific genes encoding an ω-3 and/or ω-6biosynthetic pathway into Yarrowia lipolytica containing a disruptedΔ12-desaturase (Example 8), it was necessary to confirm thefunctionality of heterologous Δ6 desaturase, elongase, Δ5 desaturase andΔ17 desaturase genes expressed in Yarrowia. This was accomplished bymeasuring the conversion efficiency of each wildtype protein in thealternate host. Specifically, a Mortierella alpina Δ5 desaturase, a M.alpina Δ6 desaturase, a Saprolegnia diclina Δ17 desaturase and a M.alpina high affinity PUFA elongase were separately expressed andscreened for activity in substrate-feeding trials.

Construction of Expression Plasmids

In general, wildtype desaturase or elongase genes were either isolatedby restriction digestion or amplified by PCR and inserted intoappropriate vectors for expression. Each PCR amplification was carriedout in a 50 μl total volume, comprising PCR buffer containing: 10 ngtemplate, 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75), 2 mMMgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (final concentration), 200 μMeach deoxyribonucleotide triphosphate, 10 pmole of each primer and 1 μlof PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.).Amplification was carried out as follows (unless otherwise specified):initial denaturation at 95° C. for 3 min, followed by 35 cycles of thefollowing: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1 min. Afinal extension cycle of 72° C. for 10 min was carried out, followed byreaction termination at 4° C.

Wild Type Mortierella alpina (Accession #AF465281) Δ6 Desaturase

The 1384 bp NcoI/NotI fragment of pCGR5 (U.S. Pat. No. 5,968,809), whichcontains the M. alpina Δ6 desaturase gene (SEQ ID NO:36), was insertedinto the NcoI/NotI sites of pY5-2 (Example 1) to generate pY54.

Wild Type Mortierella alpina (Accession #AF067654) Δ5 Desaturase

The M. alpina Δ5 desaturase gene (SEQ ID NO:38) was amplified by PCRusing oligonucleotides YL11 and YL12 (SEQ ID NOs:40 and 41) as primersand plasmid pCGR-4 (U.S. Pat. No. 6,075,183) as template. PCRamplification was carried out as described above, with the exceptionthat the elongation step was extended to 1.5 min (for cycles 1-35); The1357 bp PCR product was digested with NcoI/NotI and ligated toNcoI/NotI-digested pY5-13 (described in Example 1) to generate pYMA5pb(FIG. 6).

Wild Type Saprolegnia diclina (ATCC #56851) Δ17 Desaturase

The Wild type Δ17 desaturase gene of S. diclina was amplified fromplasmid pRSP19 (US 2003/0196217 A1) by PCR using oligonucleotides YL21A(SEQ ID NO:42) and YL22 (SEQ ID NO:43) as primers. The PCR products weredigested with NcoI/PacI and then ligated to NcoI/PacI-digested pY54(FIG. 4; described in Example 1) to generate pYSD17.

Wild Type Mortierella alpina (Accession #AX464731) High AffinityElongase

The 973 bp NotI fragment of pRPB2 (WO 00/12720), containing the codingregion of a M. alpina high affinity PUFA elongase gene (SEQ ID NO:44),was inserted into the NotI site of pY5 (described in Example 1; FIGS. 3and 4) to generate pY58.

Transformation of Yarrowia lipolytica

The plasmids pY54, pYMA5pb, pYSD17 and pY58 were transformed separatelyinto Y. lipolytica ATCC# 76982 according to the method of Chen, D. C. etal. (Appl Microbiol Biotechnol. 48(2):232-235 (1997)), and as describedin Example 2 (with the exception that a leucine auxotroph of Yarrowiawas used for transformation and transformants were selected on minimalmedia plates lacking leucine).

Determination of Percent Substrate Conversion

Single colonies of transformant Y. lipolytica containing pY54, pYMA5pb,pYSD17 or pY58 were each grown in 3 mL minimal media (20 g/L glucose,1.7 g/L yeast nitrogen base without amino acids, 1 g/L L-proline, 0.1g/L L-adenine, 0.1 g/L L-lysine, pH 6.1) at 30° C. to an OD₆₀₀˜1.0. Forsubstrate feeding, 100 μl of cells were then subcultured in 3 mL minimalmedia containing 10 μg of substrate for about 24 hr at 30° C. Cells weresubsequently collected by centrifugation and the lipids were extractedas described in the General Methods. Fatty acid methyl esters wereprepared by transesterification of the lipid extract. Percent substrateconversion was determined as: [product/(substrate+product)]*100.

Percent Substrate Conversion By M. alpina Δ6 Desaturase

The M. alpina Δ6 desaturase converts LA to GLA and/or ALA to STA. Y.lipolytica strains containing pY54 were grown as described above (nosubstrate feeding required) and lipids were analyzed. The results showedthat Yarrowia strains with pY54 converted about 30% LA to GLA.

Percent Substrate Conversion By M. alpina Δ5 Desaturase

The Δ5 desaturase from M. alpina converts DGLA to ARA and/or ETA to EPA.Y. lipolytica containing pYMA5pb was grown from a single colony,subcultured in minimal media containing 10 μg of DGLA and then subjectedto lipid analysis as described above. Yarrowia strains with pYMA5pbconverted about 30% of intracellular DGLA to ARA.

Percent Substrate Conversion By S. diclina Δ17 Desaturase

The S. diclina Δ17 desaturase converts ARA to EPA and/or DGLA to ETA. Y.lipolytica strains containing pYSD17 were grown from single colonies,subcultured in minimal media containing 10 μg of ARA and subjected tolipid analysis as described above. The results of the ARA feedingexperiments showed that Yarrowia strains with pYSD17 converted about 23%of intracellular ARA to EPA.

Percent Substrate Conversion of Wild Type M. alpina High AffinityElongase

The M. alpina high affinity PUFA elongase converts GLA to DGLA, STA toETA, and/or EPA to DPA. Y. lipolytica strains containing pY58 were grownfrom single colonies, subcultured in minimal media containing 10 μg ofGLA and subjected to lipid analysis as described above. The results ofthe GLA feeding experiments showed that Yarrowia strains with pY58converted about 30% of intracellular GLA to DGLA.

Example 6 Synthesis and Expression of a Codon-Optimized Δ17 DesaturaseGene in Yarrowia lipolytica

Based on the results of Example 5, genes encoding Δ6 desaturase,elongase and Δ5 desaturase activies were available that each enabled˜30% substrate conversion in Yarrowia lipolytica. The Δ17 desaturasefrom S. diclina, however, had a maximum conversion efficiency of only23%. Thus, a codon-optimized Δ17 desaturase gene was designed, based onthe Saprolegnia diclina DNA sequence (SEQ ID NO:35), according to theYarrowia codon usage pattern, the consensus sequence around the ‘ATG’translation initiation codon and the general rules of RNA stability(Guhaniyogi, G. and J. Brewer, Gene 265(1-2):11-23 (2001)).

In addition to modification to the translation initiation site, 127 bpof the 1077 bp coding region, comprising 117 codons, werecodon-optimized. A comparison between this codon-optimized DNA sequence(SEQ ID NO:46) and the S. diclina Δ17 desaturase gene DNA sequence (SEQID NO:35) is shown in FIG. 7, wherein nucleotides in bold textcorrespond to nucleotides that were modified in the codon-optimizedgene. None of the modifications in the codon-optimized gene changed theamino acid sequence of the encoded protein (SEQ ID NO:47).

The synthetic, codon-optimized Δ17 desaturase was suitable forexpression with other genes for PUFA biosynthesis, to test thehypothesis of whether expression in a Yarrowia lipolytica host havingits native Δ12 desaturase disrupted would result in production of “pure”ω-3 PUFAs, without co-synthesis of any ω-6 PUFAs (infra, Example 8).

Determining the Preferred Codon Usage in Yarrowia lipolytica

Approximately 100 genes of Y. lipolytica were found in the NationalCenter for Biotechnology Information public database. The coding regionsof these genes, comprising 121,167 bp, were translated by the Editseqprogram of DNAStar to the corresponding 40,389 amino acids and weretabulated to determine the Y. lipolytica codon usage profile shown inTable 9. The column titled “No.” refers to the number of times a givencodon encodes a particular amino acid in the sample of 40,389 aminoacids. The column titled “%” refers to the frequency that a given codonencodes a particular amino acid. Entries shown in bold text representthe codons favored in Yarrowia lipolytica.

TABLE 9 Codon Usage In Yarrowia lipolytica Amino Amino Condon Acid No. %Condon Acid No. % GCA Ala (A) 359 11.4 AAA Lys (K) 344 14.8 GCC Ala (A)1523 48.1 AAG Lys (K) 1987 85.2 GCG Ala (A) 256 8.1 AUG Met (M) 1002 100GCU Ala (A) 1023 32.3 UUC Phe (F) 996 61.1 AGA Arg (R) 263 13.2 UUU Phe(F) 621 38.9 AGG Arg (R) 91 4.6 CCA Pro (P) 207 9.6 CGA Arg (R) 113356.8 CCC Pro (P) 1125 52.0 CGC Arg (R) 108 5.4 CCG Pro (P) 176 8.2 CGGArg (R) 209 1.0 CCU Pro P 655 30.2 CGU Arg (R) 189 9.5 AGC Ser (S) 33511.3 AAC Ans (N) 1336 84.0 AGU Ser (S) 201 6.8 AAU Ans (N) 255 16.0 UCASer (S) 221 7.5 GAC Asp (D) 1602 66.8 UCC Ser (S) 930 31.5 GAU Asp (D)795 33.2 UCG Ser (S) 488 16.5 UGC Cys (C) 268 53.2 UCU Ser (S) 779 26.4UGU Cys (C) 236 46.8 UAA Term 38 46.9 CAA Gln (Q) 307 17.0 UAG Term 3037.0 CAG Gln (Q) 1490 83.0 UGA Term 13 16.1 GAA Glu (E) 566 23.0 ACA Thr(T) 306 12.7 GAG Glu (E) 1893 77.0 ACC Thr (T) 1245 51.6 GGA Gly (G) 85629.7 ACG Thr (T) 269 11.1 GGC Gly (G) 986 34.2 ACU Thr (T) 595 24.6 GGGGly (G) 148 5.1 UGG Trp (W) 488 100 GGU Gly (G) 893 31.0 UAC Tyr (Y) 98883.2 CAC His (H) 618 65.5 UAU Tyr (Y) 200 16.8 CAU His H 326 34.5 GUAVal (V) 118 4.2 AUA Ile (I) 42 2.1 GUC Val (V) 1052 37.3 AUC Ile (I)1106 53.7 GUG Val (V) 948 33.6 AUU Ile (I) 910 44.2 GUU Val (V) 703 24.9CUA Leu (L) 166 4.7 CUC Leu (L) 1029 29.1 CUG Leu (L) 1379 38.9 CUU Leu(L) 591 16.7 UUA Leu (L) 54 1.5 UUG Leu (L) 323 9.1

For further optimization of gene expression in Y. lipolytica, theconsensus sequence around the ‘ATG’ initiation codon of 79 genes wasexamined. In FIG. 8, the first ‘A’ of the underlined ATG translationcodon is considered to be +1. Seventy seven percent of the genesanalyzed had an ‘A’ in the −3 position, indicating a strong preferencefor ‘A’ at this position. There was also preference for ‘A’ or ‘C’ atthe −4, −2 and −1 positions, an ‘A’, ‘C’ or ‘T’ at position +5, and a‘G’ or ‘C’ at position +6. Thus, the preferred consensus sequence of thecodon-optimized translation initiation site for optimal expression ofgenes in Y. lipolytica is ‘MAMMATGNHS’ (SEQ ID NO:130), wherein thenucleic acid degeneracy code used is as follows: M=A/C; S=C/G; H=A/C/T;and N=A/C/G/T.

In Vitro Synthesis of a Codon-Optimized Gene

The method used to synthesize the codon-optimized Δ17 desaturase gene isillustrated in FIG. 9. First, eleven pairs of oligonucleotides weredesigned to extend the entire length of the codon-optimized codingregion of the S. diclina Δ17 desaturase gene (e.g., D17-1A, D17-1B,D17-2A, D17-2B, D17-3A, D17-3B, D17-4A, D17-4B, D17-5A, D17-5B, D17-6A,D17-6B, D17-7A, D17-7B, D17-8A, D17-8B, D17-9A, D17-9B, D17-10A,D17-10B, D17-11A and D17-11B, corresponding to SEQ ID NOs:48-69). Eachpair of sense (A) and anti-sense (B) oligonucleotides werecomplementary, with the exception of a 4 bp overhang at each 5′-end.Additionally, primers D17-1A, D17-4B, D17-5A, D17-8A and D17-8B alsointroduced NcoI, BglII and SalI restriction sites for subsequentsubcloning, respectively.

100 ng of each oligonucleotide was phosphorylated at 37° C. for 1 hr ina volume of 20 μl containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 10 mMDTT, 0.5 mM spermidine, 0.5 mM ATP and 10 U of T4 polynucleotide kinase.Each pair of sense and antisense oligonucleotides was mixed and annealedin a thermocycler using the following parameters: 95° C. (2 min), 85° C.(2 min), 65° C. (15 min), 37° C. (15 min), 24° C. (15 min) and 4° C. (15min). Thus, D17-1A (SEQ ID NO:48) was annealed to D17-1B (SEQ ID NO:49)to produce the double-stranded product “D17-1AB”. Similarly, D17-2A (SEQID NO:50) was annealed to D17-2B (SEQ ID NO:51) to produce thedouble-stranded product “D17-2AB”, etc.

Three separate pools of annealed, double-stranded oligonucleotides werethen ligated together, as shown below:

-   -   Pool 1: comprised D17-1AB, D17-2AB, D17-3AB and D17-4AB;    -   Pool 2: comprised D17-5AB, D17-6AB, D17-7AB and D17-8AB; and    -   Pool 3: comprised D17-9AB, D17-10AB and D17-11AB.        Each pool of annealed oligonucleotides was mixed in a volume of        20 μl with 10 U of T4 DNA ligase and the ligation reaction was        incubated overnight at 16° C.

The product of each ligation reaction was then amplified by PCR.Specifically, using the ligated “Pool 1” mixture (i.e., D17-1AB,D17-2AB, D17-3AB, and D17-4AB) as template, and oligonucleotides D17-1(SEQ ID NO:70) and D17-4R (SEQ ID NO:71) as primers, the first portionof the codon-optimized Δ17 desaturase gene was amplified by PCR. The PCRamplification was carried out in a 50 μl total volume, comprising PCRbuffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄, 20 mM Tris-HCl (pH 8.75),2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA (final concentration), 200μM each deoxyribonucleotide triphosphate, 10 pmole of each primer and 1μl of PfuTurbo DNA polymerase (Stratagene, San Diego, Calif.).Amplification was carried out as follows: initial denaturation at 95° C.for 3 min, followed by 35 cycles of the following: 95° C. for 1 min, 56°C. for 30 sec, 72° C. for 40 sec. A final extension cycle of 72° C. for10 min was carried out, followed by reaction termination at 4° C. The430 bp PCR fragment was subcloned into the pGEM-T easy vector (Promega)to generate pT17(1-4).

Using the ligated “Pool 2” mixture (i.e., D17-5AB, D17-6AB, D17-7AB andD17-8AB) as template, and oligonucleotides D17-5 (SEQ ID NO:72) andD17-8D (SEQ ID NO:73) as primers, the second portion of thecodon-optimized Δ17 desaturase gene was amplified similarly by PCR andcloned into pGEM-T-easy vector to generate pT17(5-8). Finally, using the“Pool 3” ligation mixture (i.e., D17-9AB, D17-10AB and D17-11AB) astemplate, and oligonucleotides D17-8U (SEQ ID NO:74) and D17-11 (SEQ IDNO:75) as primers, the third portion of the codon-optimized Δ17desaturase gene was amplified similarly by PCR and cloned intopGEM-T-easy vector to generate pT17(9-11).

E. coli was transformed separately with pT17(1-4), pT17(5-8) andpT17(9-11) and the plasmid DNA was isolated from ampicillin-resistanttransformants. Plasmid DNA was purified and digested with theappropriate restriction endonucleases to liberate the 420 bp NcoI/BglIIfragment of pT17(14), the 400 bp BglII/SalI fragment of pT17(5-8) andthe 300 bp SalI/NotI fragment of pT17(9-11). These fragments were thencombined, ligated together and used as template for amplification of theentire synthetic codon-optimized Δ17 desaturase gene using D17-1 (SEQ IDNO:70) and D17-11 (SEQ ID NO:75) as primers. The PCR amplification wascarried out in a 50 μl total volume, using the conditions describedabove for each portion of the Δ17 desaturase gene and the thermocyclingprogram as follows: initial denaturation at 95° C. for 3 min, followedby 35 cycles of the following: 95° C. for 1 min, 56° C. for 30 sec, 72°C. for 1.1 min. A final extension cycle of 72° C. for 10 min was carriedout, followed by reaction termination at 4° C. This generated a 1.1 kBPCR product.

Construction of Plasmid pYSD17S Containing the Codon-Optimized Δ17Desaturase

The 1.1 kB PCR product comprising the entire synthetic Δ17-desaturasewas digested with NcoI/NotI and subcloned into NcoI/NotI-digested pY5-13(Example 1) to generate pYSD17S (FIG. 10A).

As an additional “control”, to compare the efficiency of the wild typeand synthetic genes in Yarrowia, the AT-rich PacI site in pYSD17(comprising the wild-type gene; described in Example 5) was eliminatedby site-directed mutagenesis using YL53 (SEQ ID NO:76) and YL54 (SEQ IDNO:77) as primers to generate pYSD17M (FIG. 10B).

Transformation of Yarrowia lipolytica with the Codon-Optimized Δ17Desaturase Gene

Plasmids containing the wildtype and codon-optimized Δ17 desaturase weretransformed separately into Y. lipolytica ATCC #76982 according to themethods described above in Example 5. Using this technique,transformants were obtained that contained the following plasmids:

TABLE 10 Summary Of Plasmids In Transformant Yarrowia PlasmidDescription pYSD17 wildtype Δ17 desaturase pYSD17M wildtype Δ17desaturase, minus AT-rich PacI site pYSD17S codon-optimized Δ17desaturasePercent Substrate Conversion with the Codon-Optimized Δ17 DesaturaseGene

Δ17 desaturase converts ARA to EPA (see FIG. 2). The percent substrateconversion ([product]/[substrate+product]*100) of the wildtype andcodon-optimized Δ17 desaturase genes was determined in Yarrowialipolytica containing each alternate plasmid construct, using themethodology described in Example 5.

The results of the ARA feeding experiments showed that Yarrowia strainswith control plasmids pYSD17 or pYSD17M converted about 23% ofintracellular ARA to EPA (FIG. 11A) while those containing thecodon-optimized Δ17 desaturase gene within pYSD17S converted about 45%of intracellular ARA to EPA (FIG. 11B). Thus, Yarrowia containing thecodon-optimized Δ17 desaturase converted about 2-fold more ARA than thestrains containing the wild type S. diclina gene.

Example 7 Construction of Plasmids Suitable for the CoordinateExpression of Multiple Omega Fatty Acid Biosynthesis Genes in Yarrowialipolytica

A variety of expression plasmids were constructed to produce a constructcomprising a Δ6 desaturase, elongase, Δ5 desaturase, and Δ17 desaturasethat would be suitable to integrate into the Y. lipolytica genome.Expression of this construct was necessary to test the hypothesize that“pure” ω-3 PUFAs, without co-synthesis of any ω-6 PUFAs, could beproduced in a Y. lipolytica host containing a disrupted native Δ12desaturase (infra, Example 8).

Construction of Plasmid pY24

Plasmid pY24 (FIG. 12) was a parent vector for construction ofexpression cassettes suitable for integration into the genome ofYarrowia lipolytica. pY24 was constructed as follows:

Using oligonucleotides KU5 and KU3 (SEQ ID NOs:78 and 79) as primers andYarrowia genomic DNA as template, a 1.7 kB DNA fragment (SEQ ID NO:80)containing the Yarrowia URA3 gene was PCR amplified. The PCRamplification was carried out in a 50 μl total volume containing: 100 ngYarrowia genomic DNA, PCR buffer containing 10 mM KCl, 10 mM (NH₄)₂SO₄,20 mM Tris-HCl (pH 8.75), 2 mM MgSO₄, 0.1% Triton X-100, 100 μg/mL BSA(final concentration), 200 μM each deoxyribonucleotide triphosphate, 10pmole of each primer and 1 μl of PfuTurbo DNA polymerase (Stratagene,San Diego, Calif.). Amplification was carried out as follows: initialdenaturation at 95° C. for 3 min, followed by 35 cycles of thefollowing: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 2 min. Afinal extension cycle of 72° C. for 10 min was carried out, followed byreaction termination at 4° C. The PCR product was inserted into pGEM-Teasy vector (Promega, Madison, Wis.) to generate pGYUM.

Using oligonucleotides KI5 and KI3 (SEQ ID NOs:82 and 83), a 1.1 kB DNAfragment (SEQ ID NO:84) containing the conjugase gene (or “imp H8”) ofImpatients balsama (clone ids.pk0001.h8; E. I. du Pont de Nemours andCompany, Inc., Wilmington, Del.) was PCR amplified. The PCRamplification was carried out using the components described above, withthe exception that 10 ng plasmid DNA of ids.pk0001.h8 was used astemplate. Amplification was carried out as follows: initial denaturationat 95° C. for 3 min, followed by 35 cycles of the following: 95° C. for1.5 min, 56° C. for 30 sec, 72° C. for 1.2 min. A final extension cycleof 72° C. for 10 min was carried out, followed by reaction terminationat 4° C. The PCR products were digested with NotI, and then insertedinto the NotI site of pY5 (FIG. 3) to generate pY9.

Using oligonucleotides KTI5 and KTI3 (SEQ ID NOs:86 and 87), a 1.7 kBDNA fragment (SEQ ID NO:88) containing the TEF::IMP H8::XPR chimericgene of pY9 was PCR amplified. The PCR amplification was carried out asdescribed above, with the exception that 10 ng plasmid DNA of pGYUM wasused as template. Amplification was carried out as follows: initialdenaturation at 95° C. for 3 min, followed by 35 cycles of thefollowing: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 2 min. Afinal extension cycle of 72° C. for 10 min was carried out, followed byreaction termination at 4° C. The PCR products were inserted intoPCR-Script (Stratagene) to generate pY9R. The 1.7 kB Xho/EcoRV fragmentof pY9R was exchanged with the XhoI/EcoRV fragment of pGYUM to generatepY21.

Using oligonucleotides KH5 and KH3 (SEQ ID NOs:90 and 91) as primers andgenomic DNA of KS65 as template, a 1 kB DNA fragment (SEQ ID NO:92)containing the E. coli hygromycin resistance gene (“HPT”; Kaster, K. R.,et al., Nucleic Acids Res. 11:6895-6911 (1983)) was PCR amplified. ThePCR amplification was carried out in a 50 μl total volume using thecomponents described above, with the exception that 10 ng plasmid DNA ofids.pk0001.h8 was used as template. Amplification was carried out asfollows: initial denaturation at 95° C. for 3 min, followed by 35 cyclesof the following: 95° C. for 1 min, 56° C. for 30 sec, 72° C. for 1.2min. A final extension cycle of 72° C. for 10 min was carried out,followed by reaction termination at 4° C. The PCR products were digestedwith NotI and then inserted into the NotI site of pY5 (FIG. 3) togenerate pTHPT-1.

Using oligonucleotides KTH5 and KTH3 (SEQ ID NOs:94 and 95) as primersand pTHPT-1 plasmid DNA as template, a 1.6 kB DNA fragment (SEQ IDNO:96) containing the TEF::HPT::XPR fusion gene was amplified asdescribed above. The PCR products were digested with BglII and theninserted into pY21 to generate pY24.

Construction of pY24-4

Plasmid pY24 (FIG. 12) was used for construction of expression cassettessuitable for integration into the Y. lipolytica genome. The 401 bp of5′-sequence (SEQ ID NO:98) and 568 bp of 3′-sequence (SEQ ID NO:99) fromthe Yarrowia lipolytica URA3 gene in pY24 plasmid were used to directintegration of expression cassettes into the Ura loci of the Yarrowiagenome. Two chimeric genes (TEF::HPT::XPR and TEF::IMP H8::XPR) werefirst removed from pY24 by digestion with BamHI and self-ligation togenerate pY24-1. PacI and BsiWI sites were introduced into pY24-1 bysite-directed mutagenesis using YL63 and YL64 (SEQ ID NOs:100 and 101)and YL65 and YL66 (SEQ ID NOs:102 and 103) primer pairs, respectively,to generate pY24-4.

Construction of an Integration Vector for Expression of Δ5 Desaturase

The 4261 bp PacI/BsiWI fragment of pYMA5pb (comprising the M. alpina Δ5desaturase gene; described in Example 5) was ligated into the PacI/BsiWIsites of pY24-4 (FIG. 12) to generate pYZM5 (FIG. 6). HindIII and ClaIsites were introduced into pYZM5 by site-directed mutagenesis usingprimer pairs YL81 and YL82 (SEQ ID NOs:104 and 105) and YL83 and YL84(SEQ ID NOs:106 and 107), respectively, to generate pYZM5CH (FIG. 6). APmeI site was introduced into pYZM5CH by site-directed mutagenesis usingYL105 and YL106 (SEQ ID NOs:108 and 109) as primers to generatepYZM5CHPP. An AscI site was introduced into pYZM5CHPP by site-directedmutagenesis using YL119 and YL120 (SEQ ID NOs:110 and 111) as primers togenerate pYZM5CHPPA (FIG. 6).

To optimize the integration vector, 440 bp of 5′-non-coding DNA sequenceupstream from the Yarrowia lipolytica URA3 gene (SEQ ID NO:114) wasamplified by PCR using YL121 and YL122 (SEQ ID NOs:112 and 113) asprimers. The PCR product was digested with AscI and BsiWI and thenexchanged with the AscI/BsiWI fragment of pYZM5CHPPA (FIGS. 6 and 13) togenerate pYZM5UPA (FIG. 13). An AscI site was introduced into pYZM5UPAby site-directed mutagenesis using oligonucleotides YL114 and YL115 (SEQID NOs:115 and 116) to generate pYZV5. In order to reduce the size ofthe 3′-non-coding region of the URA3 gene in pYZV5, a second PacI sitewas introduced into the middle of this region by site-directedmutagenesis using oligonucleotides YL114 and YL115 (described above) togenerate pYZV5P. The PacI fragment of pYZV5P was excised by digestionwith PacI and religation to generate pYZV16 (FIG. 13). Digestion ofpYZV16 with AscI liberates a 5.2 kB DNA fragment (SEQ ID NO:117)suitable for integration and expression of the Δ5 desaturase gene(“MAD5”) in the Y. lipolytica genome.

Construction of an Integration Vector for Expression of the HighAffinity Elongase and Δ5 Desaturase

BsiWI and HindIII sites were introduced into pY58 (containing the codingregion of the M. alpina high affinity PUFA elongase; described inExample 5) by site-directed mutagenesis using YL61 and YL62 (SEQ IDNOs:17 and 18) and YL69 and YL70 (SEQ ID NOs:118 and 119) primer pairs,respectively, to generate pY58BH (FIG. 14; elongase gene labeled as“EL”). The 1.7 kB BsiWI/HindIII fragment of pY58BH, which contains theTEF::EL::XPR chimeric gene, was ligated into the BsiWI/HindIII site ofpYZM5CHPP (construction described in FIG. 6) to generate pYZM5EL (FIG.14). This plasmid is suitable for integration and coordinate expressionof the M. alpina Δ5 desaturase and high affinity PUFA elongase genes inY. lipolytica.

Construction of an Integration Vector for Expression of the Δ6Desaturase, High Affinity Elongase and Δ5 Desaturase

PacI and ClaI sites were introduced into pY54 (containing the M. alpinaΔ6 desaturase; described in Example 5) by site-directed mutagenesisusing YL77 and YL78 (SEQ ID NOs:120 and 121) and YL79A and YL80A (SEQ IDNOs:122 and 123) primer pairs, respectively, to generate pY54PC (FIG.14; Δ6 desaturase gene labeled as “MAD6”). The 2 kB ClaI/PacI DNAfragment of pY54PC, which contains the TEF::MAD6::XPR chimeric gene, wasligated into the ClaI/PacI sites of pYZM5EL to generate pYZM5EL6 (FIG.14). This plasmid is suitable for integration and coordinate expressionof the M. alpina Δ6 desaturase, Δ5 desaturase and high affinity PUFAelongase genes in the Y. lipolytica genome.

Construction of a DNA Fragment Suitable for Integration into theYarrowia Genome, for Expression of the Δ6 Desaturase, PUFA Elongase andΔ5 Desaturase

The plasmid pYZV16 (construction described in FIG. 13) was used forconstruction of plasmids containing multiple expression cassettes.

First, the 3.5 kB BsiWI/PacI fragment of pYZV16 was ligated to the 7.9kB BsiWI/PacI fragment of pYZM5EL6 (construction described in FIG. 14)to generate pYZV5EL6 (FIG. 15). Digestion of pYZV5EL6 with AscIliberates a 8.9 kB DNA fragment (SEQ ID NO:124) suitable for integrationand coordinate expression of the Δ6 desaturase, PUFA elongase and Δ5desaturase genes in the Y. lipolytica genome.

Construction of a DNA Fragment Suitable for Integration into theYarrowia Genome, for Expression of the Δ6 Desaturase, PUFA Elongase, Δ5Desaturase and Δ17 Desaturase

A synthetic S. diclina Δ17 desaturase gene was inserted into theNcoI/NotI sites of pY5-13 to generate pYSD17S (FIG. 1A). ClaI and PmeIsites were introduced into pYSD17S by site-directed mutagenesis usingYL101 and YL102 (SEQ ID NOs:125 and 126) and YL103 and YL104 (SEQ IDNOs:127 and 128) primer pairs, respectively, to generate pYSD17SPC (FIG.15).

The 347 bp ClaI/PmeI fragment of pYZV5EL6 (FIG. 15) was exchanged withthe 1760 bp ClaI/PmeI fragment from pYSD17SPC containing the Δ17desaturase expression cassette to generate pYZV5E6/17. Digestion ofpYZV5E6/17 with AscI liberates a 10.3 kB DNA fragment (SEQ ID NO:129)suitable for integration and coordinate expression of the Δ6 desaturase,PUFA elongase, Δ5 desaturase and Δ17 desaturase genes in the Y.lipolytica genome.

Example 8 Use of Δ12 Desaturase Disrupted Strains for the Production ofPure Omega-3 Fatty Acids by Substrate Feeding

The present Example describes the utility of a Δ12 desaturase-disruptedYarrowia lipolytica host strain containing appropriate heterologousgenes (e.g., a Δ6 desaturase, elongase, Δ5 desaturase, Δ17 desaturase,as described in Example 7) for the production of ω-3 PUFAs, withoutco-synthesis of any ω-6 PUFAs. Feeding studies were performed with ALAas the substrate. The results demonstrate that it is possible to produceω-3 PUFAs in the absence of ω-6 PUFAs.

Feeding Studies

Wildtype Yarrowia lipolytica ATCC #76982 was transformed with anintegrating 10.3 kB DNA fragment (SEQ ID NO:129) containing a Δ6desaturase, PUFA elongase, Δ5 desaturase and Δ17 desaturase (fromExample 7). This resulted in creation of strain “WT+4G”. Then, the Δ12desaturase was disrupted in strain WT+4G, as described in Example 2.This resulted in creation of strain “D12KO+4G”.

Cells from each of the four strains listed below in Table 11 (100 μl)were grown in 3 mL minimal media containing no substrate addition, 10 μgof LA, 10 ug ALA, or 5 ug each of LA and ALA for about 24 hr at 30° C.

TABLE 11 Description Of Strains Tested In The Feeding Studies StrainDesignation Strain Description Example WT wild-type Yarrowia lipolyticaATCC #76982 — WT + 4G wild-type Yarrowia lipolytica, containing a 8 Δ6desaturase, PUFA elongase, Δ5 desaturase and Δ17-desaturase D12KO Δ12desaturase-disrupted Yarrowia 2 lipolytica D12KO + 4G Δ12desaturase-disrupted Yarrowia 8 lipolytica, containing a Δ6 desaturase,PUFA elongase, Δ5 desaturase and Δ17 desaturase

Fatty acid composition was determined by direct transesterification, asdescribed in the General Methods. The fatty acid profile of each of thestrains grown with no substrate addition, 10 μg of LA, 10 ug ALA, or 5ug each of LA and ALA are shown below in Table 12. Fatty acids areidentified as 16:0, 16:1, 18:0, 18:1 (oleic acid), 18:2 (LA), GLA, DGLA,ALA, and STA. The composition of each is presented as a % of the totalfatty acids.

TABLE 12 Fatty Acid Composition (% Of Total Fatty Acids) Strain FA feed16:0 16:1 18:0 18:1 18:2 GLA DGLA ALA STA WT None 10 6 8 50 21 nd nd ndnd WT LA 11 3 6 30 47 nd nd nd nd WT ALA 9 3 4 29 5 nd nd 48 nd D12KONone 10 7 8 68 nd nd nd nd nd D12KO LA 9 4 5 26 53 nd nd nd nd D12KO ALA10 4 6 41 nd nd nd 35 nd WT + 4G None 11 6 7 57 6 5 0.9 nd nd WT + 4G LA11 3 6 32 31 9 1.0 nd nd WT + 4G ALA 9 3 5 31 2 1 0.2 40 4 WT + 4G LA +A 6 1 2 10 33 4 0.3 39 2 D12KO + 4G None 9 6 8 69 nd nd nd nd nd D12KO +4G LA 8 2 6 24 45 10 1.0 nd nd D12KO + 4G ALA 8 5 5 45 nd nd nd 27 4D12KO + 4G LA + A 7 2 4 14 26 4 0.3 37 3 *nd = not detectable

The results showed that feeding ALA to D12 KO cells resulted in theproduction of only ω-3 fatty acids (i.e., ALA and STA), withoutbiosynthesis of any ω-6 fatty acids (i.e., GLA or DGLA).

1. A method for the production of linoleic acid comprising: a) providinga microbial host cell, wherein the microbial host cell is Yarrowia,comprising: (i) a chimeric gene encoding a Δ12 desaturase polypeptideselected from the group consisting of: (a) an isolated nucleic acidmolecule encoding a polypeptide having Δ12 desaturase activity whereinthe polypeptide has at least 90% sequence identity when compared to anamino acid sequence as set forth in SEQ ID NO:24, and (b) an isolatednucleic acid molecule encoding a polypeptide having Δ12 desaturaseactivity, wherein the nucleic acid molecule hybridizes with a nucleotidesequence as set forth in SEQ ID NO:23 under the following hybridizationconditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDSfollowed by 0.1×SSC, 0.1% SDS; and (ii) a source of desaturase substrateconsisting of oleic acid; b) growing the microbial host cell of step (a)under conditions wherein the gene encoding a Δ12 desaturase polypeptideis expressed and the oleic acid is converted to linoleic acid; and c)optionally recovering the linoleic acid of step (b).
 2. A method formodulating the biosynthesis of ω-3 fatty acids in an oleaginous yeastcell comprising: a) providing an oleaginous yeast cell, comprising afunctional ω-3 fatty acid biosynthetic pathway; b) over-expressing a Δ12desaturase gene in the oleaginous yeast of (a), wherein said Δ12desaturase gene is selected from the group consisting of: (i) anisolated nucleic acid molecule encoding a polypeptide having Δ12desaturase activity wherein the polypeptide has at least 90% sequenceidentity when compared to an amino acid sequence as set forth in SEQ IDNO:24, and (ii) an isolated nucleic acid molecule encoding a polypeptidehaving Δ12 desaturase activity, wherein the nucleic acid moleculehybridizes with a nucleotide sequence as set forth in SEQ ID NO:23 underthe following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. andwashed with 2×SSC, 0.1% SDS followed by 0.1×SSC, 0.1% SDS; whereby thebiosynthesis of ω-3 fatty acids is modulated.
 3. The method according toclaim 2 wherein the Δ12 desaturase gene is over-expressed on a multicopyplasmid.
 4. The method according to claim 2 wherein the Δ12 desaturasegene is operably linked to an inducible or regulated promoter.